D-amino acid compounds for liver disease

ABSTRACT

Provided herein are compounds, compositions and methods for the treatment of liver disease and conditions, including HCV infections. In certain embodiments, compounds and compositions of nucleoside derivatives are disclosed, which can be administered either alone or in combination with other anti-viral agents.

FIELD

Provided herein are compounds, methods and pharmaceutical compositionsfor use in treatment of liver diseases and conditions, including viralinfections such as hepatitis C virus infections in hosts in needthereof. In certain embodiments, D-amino acids linked to therapeuticnucleoside analogs are provided which display remarkable efficacy andbioavailability for the treatment of, for example, HCV infection in ahuman.

BACKGROUND

The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide. (Boyer, N. et al., J. Hepatol. 32:98-112, 2000). HCVcauses a slow growing viral infection and is the major cause ofcirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon,B. R., Scientific American, October: 80-85, 1999; Boyer, N. et al., J.Hepatol. 32:98-112, 2000). It is estimated there are about 130-170million people with chronic hepatitis C virus infection, and there areabout 350,000 deaths from hepatitis C-related liver diseases each year(Hepatitis C Fact Sheet, World Health Organization Fact Sheet No. 164,June 2011). Cirrhosis caused by chronic hepatitis C infection accountsfor 8,000-12,000 deaths per year in the United States, and HCV infectionis the leading indication for liver transplantation.

HCV infection becomes chronic in about 75% of cases, with many patientsinitially being asymptomatic. The first symptoms of HCV infection areoften those of chronic liver disease. About 20 to 30% of patients withchronic hepatitis due to HCV develop cirrhosis, although this may takedecades. Development of cirrhosis due to HCV also increases the risk ofhepatocellular cancer (The Merck Manual Online, Chronic Hepatitis,available atwww.merckmanuals.com/professional/hepatic_and_biliary_disorders/hepatitis/chronic_hepatitis.html,last revision February 2007).

In light of the fact that HCV infection has reached epidemic levelsworldwide, and has tragic effects on the infected patient, there remainsa strong need to provide new effective pharmaceutical agents to treathepatitis C that have low toxicity to the host. Further, given therising threat of other flaviviridae infections, there remains a strongneed to provide new effective pharmaceutical agents that have lowtoxicity to the host. Therefore, there is a continuing need foreffective treatments of flavivirus infections and HCV infections.

SUMMARY

Provided herein are compounds useful for treatment of liver diseases andconditions, for example, for the treatment of flavivirus infections suchas HCV infections. The compounds comprise D-amino acids linked totherapeutic moieties. In certain embodiments the D-amino acid compoundsdisplay high tissue levels of active species, remarkable efficacy, orbioavailability, or all, for the treatment of, for example, liverdisease and conditions in a human in need thereof. Some of the compoundsare based, in part, on the discovery that the active component ofcertain therapeutic moieties linked to D-amino acids can accumulatefavorably in liver cells when the compounds are administered tosubjects.

In certain embodiments, the compounds provided herein are useful in theprevention and treatment of Flaviviridae infections and other relatedconditions such as anti-Flaviviridae antibody positive andFlaviviridae-positive conditions, chronic liver inflammation caused byHCV, cirrhosis, fibrosis, acute hepatitis, fulminant hepatitis, chronicpersistent hepatitis and fatigue. These compounds or formulations canalso be used prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-Flaviviridae antibody orFlaviviridae-antigen positive or who have been exposed to aFlaviviridae. In particular embodiments, the Flaviviridae is hepatitisC. In certain embodiments, the compounds are used to treat any virusthat replicates through an RNA-dependent RNA polymerase.

A method for the treatment of a Flaviviridae infection in a host,including a human, is also provided that includes administering aneffective amount of a compound provided herein, administered eitheralone or in combination or alternation with another anti-Flaviviridaeagent, optionally in a pharmaceutically acceptable carrier.

In certain embodiments, provided herein are compounds according toformula (2001):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof, wherein: Base is anucleobase; A is S or O; W is S or O; X is a D-amino acid residue, or anester thereof; Y is hydrogen, —OR¹, —SR¹, or —NR¹R²; R^(b1) is alkyl,cycloalkyl, —H, azido, cyano, or halogen; R^(b2) is —OH, —Cl, —F, —H,azido, cyano, amino, or alkoxyl, or, in the alternative, R^(b1) andR^(b2), along with the carbon atom to which they are attached, join toform a three-membered carbocyclic or heterocyclic ring; R^(c) is —H or—OH, or, in the alternative, Y and R^(c) join to form a six-memberedheterocyclic ring wherein Y and R^(c) together represent a singledivalent —O—; R^(d) is —H, —F, azido, or allenyl; or, in thealternative, R^(b2) and R^(d) join to form alkylene or substitutedalkylene; R^(e) is —H or alkyl; each R¹ is independently alkyl,cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, substitutedalkyl or hydantoinylalkyl; and each R² is independently hydrogen oralkyl.

In one aspect, the compounds provided herein are provided oradministered in combination with a second therapeutic agent, such as oneuseful for the treatment or prevention of HCV infections. Exemplarysecond therapeutic agents are provided in detail elsewhere herein.

In another aspect, provided herein are pharmaceutical compositions,single unit dosage forms, and kits suitable for use in treating orpreventing disorders such as HCV infections which comprise atherapeutically or prophylactically effective amount of a compoundprovided herein and a therapeutically or prophylactically effectiveamount of a second therapeutic agent such as one useful for thetreatment or prevention of HCV infections.

In certain embodiments, a method of treatment of a liver disease ordisorder is provided comprising administering to an individual in needthereof a treatment effective amount of a compound provided herein.

Flaviviridae which can be treated are, e.g., discussed generally inFields Virology, Fifth Ed., Editors: Knipe, D. M., and Howley, P. M.,Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapters33-35, 2006. In a particular embodiment of the invention, theFlaviviridae is HCV. In an alternate embodiment, the Flaviviridae is aflavivirus or pestivirus. In certain embodiments, the Flaviviridae canbe from any class of Flaviviridae. In certain embodiments, theFlaviviridae is a mammalian tick-borne virus. In certain embodiments,the Flaviviridae is a seabird tick-borne virus. In certain embodiments,the Flaviviridae is a mosquito-borne virus. In certain embodiments, theFlaviviridae is an Aroa virus. In certain embodiments, the Flaviviridaeis a Dengue virus. In certain embodiments, the Flaviviridae is aJapanese encephalitis virus. In certain embodiments, the Flaviviridae isa Kokobera virus. In certain embodiments, the Flaviviridae is a Ntayavirus. In certain embodiments, the Flaviviridae is a Spondweni virus. Incertain embodiments, the Flaviviridae is a Yellow fever virus. Incertain embodiments, the Flaviviridae is a Entebbe virus. In certainembodiments, the Flaviviridae is a Modoc virus. In certain embodiments,the Flaviviridae is a Rio Bravo virus.

Specific flaviviruses include, without limitation: Absettarov, Aedes,Alfuy, Alkhurma, Apoi, Aroa, Bagaza, Banzi, Bukalasa bat, Bouboui,Bussuquara, Cacipacore, Calbertado, Carey Island, Cell fusing agent,Cowbone Ridge, Culex, Dakar bat, Dengue 1, Dengue 2, Dengue 3, Dengue 4,Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr, Ilheus, Israelturkey meningoencephalitis, Japanese encephalitis, Jugra, Jutiapa,Kadam, Kamiti River, Karshi, Kedougou, Kokobera, Koutango, Kumlinge,Kunjin, Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc,Montana myotis leukoencephalitis, Murray valley encephalitis, Nakiwogo,Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat,Powassan, Quang Binh, Rio Bravo, Rocio, Royal Farm, Russianspring-summer encephalitis, Saboya, St. Louis encephalitis, Sal Vieja,San Perlita, Saumarez Reef, Sepik, Sokuluk, Spondweni, Stratford,Tembusu, Tick-borne encephalitis, Turkish sheep encephalitis, Tyuleniy,Uganda S, Usutu, Wesselsbron, West Nile, Yaounde, Yellow fever, Yokose,and Zika.

Pestiviruses which can be treated are discussed generally in FieldsVirology, Fifth Ed., Editors: Knipe, D. M., and Howley, P. M.,Lippincott Williams & Wilkins Publishers, Philadelphia, Pa., Chapters33-35, 2006. Specific pestiviruses include, without limitation: bovineviral diarrhea virus (“BVDV”), classical swine fever virus (“CSFV,” alsocalled hog cholera virus), and border disease virus (“BDV”).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are compounds, compositions and methods useful fortreating liver disorders such as HCV infection in a subject. Furtherprovided are dosage forms useful for such methods.

Definitions

When referring to the compounds provided herein, the following termshave the following meanings unless indicated otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill in the art.In the event that there is a plurality of definitions for a term herein,those in this section prevail unless stated otherwise.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight or branched hydrocarbon. In certain embodiments,the alkyl group is a primary, secondary, or tertiary hydrocarbon. Incertain embodiments, the alkyl group includes one to ten carbon atoms,i.e., C₁ to C₁₀ alkyl. In certain embodiments, the alkyl group isselected from the group consisting of methyl, CF₃, CCl₃, CFCl₂, CF₂Cl,ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl, butyl, isobutyl, secbutyl,t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term includes bothsubstituted and unsubstituted alkyl groups, including halogenated alkylgroups. In certain embodiments, the alkyl group is a fluorinated alkylgroup. Non-limiting examples of moieties with which the alkyl group canbe substituted are selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl,” as used herein, and unless otherwise specified,refers to a saturated straight or branched hydrocarbon having one to sixcarbon atoms, i.e., C₁ to C₆ alkyl. In certain embodiments, the loweralkyl group is a primary, secondary, or tertiary hydrocarbon. The termincludes both substituted and unsubstituted moieties.

The term “upper alkyl,” as used herein, and unless otherwise specified,refers to a saturated straight or branched hydrocarbon having seven tothirty carbon atoms, i.e., C₇ to C₃₀ alkyl. In certain embodiments, theupper alkyl group is a primary, secondary, or tertiary hydrocarbon. Theterm includes both substituted and unsubstituted moieties.

The term “cycloalkyl,” as used herein, unless otherwise specified,refers to a saturated cyclic hydrocarbon. In certain embodiments, thecycloalkyl group may be a saturated, and/or bridged, and/or non-bridged,and/or a fused bicyclic group. In certain embodiments, the cycloalkylgroup includes three to ten carbon atoms, i.e., C₃ to C₁₀ cycloalkyl. Insome embodiments, the cycloalkyl has from 3 to 15 (C₃₋₁₅), from 3 to 10(C₃₋₁₀), or from 3 to 7 (C₃₋₇) carbon atoms. In certain embodiments, thecycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, decalinyl or adamantyl. The term includes bothsubstituted and unsubstituted cycloalkyl groups, including halogenatedcycloalkyl groups. In certain embodiments, the cycloalkyl group is afluorinated cycloalkyl group. Non-limiting examples of moieties withwhich the cycloalkyl group can be substituted are selected from thegroup consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy,nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, orphosphonate, either unprotected, or protected as necessary.

“Cyclopropylene,” as used herein, refers to a divalent cyclopropanegroup. In certain embodiments, a cyclopropylene group is of formula

“Oxiranylene,” as used herein, refers to a divalent oxirane group. Incertain embodiments, a oxiranylene group is of formula

“Alkylene” refers to divalent saturated aliphatic hydrocarbon groupsparticularly having from one to eleven carbon atoms which can bestraight-chained or branched. In certain embodiments, the alkylene groupcontains 1 to 10 carbon atoms. The term includes both substituted andunsubstituted moieties. This term is exemplified by groups such asmethylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like. The term includes groupshaving more than one double bond, such as allenes comprising anallenylene (>C═C═C<) or allenyl (>C═C═CH₂) group. The term includeshalogenated alkylene groups. In certain embodiments, the alkylene groupis a fluorinated alkylene group. Non-limiting examples of moieties withwhich the alkylene group can be substituted are selected from the groupconsisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,carbonyl, sulfanyl, amino, alkylamino, alkylaryl, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, and phosphonate, either unprotected, or protected asnecessary.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbongroups, in certain embodiment, having up to about 11 carbon atoms, from2 to 8 carbon atoms, or from 2 to 6 carbon atoms, which can bestraight-chained or branched and having at least 1 or from 1 to 2 sitesof olefinic unsaturation. The term includes both substituted andunsubstituted moieties. Exemplary alkenyl groups include ethenyl (i.e.,vinyl, or —CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl (—C(CH₃)═CH₂),and the like. The term includes halogenated alkenyl groups. In certainembodiments, the alkenyl group is a fluorinated alkenyl group.Non-limiting examples of moieties with which the alkenyl group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

The term “cycloalkenyl,” as used herein, unless otherwise specified,refers to an unsaturated cyclic hydrocarbon. In certain embodiments,cycloalkenyl refers to mono- or multicyclic ring systems that include atleast one double bond. In certain embodiments, the cycloalkenyl groupmay be a bridged, non-bridged, and/or a fused bicyclic group. In certainembodiments, the cycloalkyl group includes three to ten carbon atoms,i.e., C₃ to C₁₀ cycloalkyl. In some embodiments, the cycloalkenyl hasfrom 3 to 7 (C₃₋₁₀), or from 4 to 7 (C₃₋₇) carbon atoms. The termincludes both substituted and unsubstituted cycloalkenyl groups,including halogenated cycloalkenyl groups. In certain embodiments, thecycloalkenyl group is a fluorinated cycloalkenyl group. Non-limitingexamples of moieties with which the cycloalkenyl group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

“Alkenylene” refers to divalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms orfrom 2 to 6 carbon atoms which can be straight-chained or branched andhaving at least 1 or from 1 to 2 sites of olefinic unsaturation. Thisterm is exemplified by groups such as ethenylene (—CH═CH—), thepropenylene isomers (e.g., —CH═CHCH₂— and —C(CH₃)═CH— and —CH═C(CH₃)—)and the like. The term includes both substituted and unsubstitutedalkenylene groups, including halogenated alkenylene groups. In certainembodiments, the alkenylene group is a fluorinated alkenylene group.Non-limiting examples of moieties with which the alkenylene group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

“Alkynyl” refers to acetylenically unsaturated hydrocarbon groups, incertain embodiments, having up to about 11 carbon atoms or from 2 to 6carbon atoms which can be straight-chained or branched and having atleast 1 or from 1 to 2 sites of alkynyl unsaturation. Non-limitingexamples of alkynyl groups include acetylenic, ethynyl (—C≡CH),propargyl (—CH₂C≡CH), and the like. The term includes both substitutedand unsubstituted alkynyl groups, including halogenated alkynyl groups.In certain embodiments, the alkynyl group is a fluorinated alkynylgroup. Non-limiting examples of moieties with which the alkynyl groupcan be substituted are selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary.

The term “aryl,” as used herein, and unless otherwise specified, refersto phenyl, biphenyl or naphthyl. The term includes both substituted andunsubstituted moieties. An aryl group can be substituted with anydescribed moiety, including, but not limited to, one or more moietiesselected from the group consisting of halogen (fluoro, chloro, bromo oriodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991.

“Alkoxy” refers to the group —OR′ where R′ is alkyl or cycloalkyl.Alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

The term “heterocyclylalkyl” refers to a radical -alkyl-heterocyclyl,where alkyl and heterocyclyl are as defined herein.

The term “alkylcarbonylthioalkyl” refers to a radical-alkyl-S—C(O)-alkyl, where alkyl is as defined herein.

The term “alkoxycarbonylalkyl” refers to a radical -alkyl-C(O)-alkoxy,where alkyl and alkoxy are as defined herein.

The term “arylalkoxycarbonylalkyl” refers to a radical-alkyl-C(O)-alkoxy-aryl, where alkyl, alkoxy and aryl are as definedherein.

The term “alkylcarbonylalkoxy(arylalkyl)” refers to a radical-alkoxy(-alkyl-aryl)-C(O)-alkyl, where alkyl, alkoxy and aryl are asdefined herein.

The term “(alkoxycarbonyl)(alkoxycarbonylamino)alkyl” refers to aradical -alkyl(-carbonyl-alkoxy)(-amino-carbonyl-alkoxy), where alkyl,carbonyl, alkoxy, and amino are as defined herein.

The term “cycloalkylcarbonylalkoxyl” refers to a radical-alkoxyl-C(O)-cycloalkyl, where alkoxyl and cycloalkyl are as definedherein.

The term “alkoxycarbonylaminoalkylcarbonylthioalkyl” refers to a radical-alkyl-S—C(O)—NH-alkyl-C(O)-alkoxy or-alkyl-S—C(O)-alkyl-NH—C(O)-alkoxy, where alkyl and alkoxy are asdefined herein.

The term “hydroxylalkylcarbonylthioalkyl” refers to a radical-alkyl-S—C(O)— alkyl-OH, where alkyl is as defined herein.

The term “aminoalkylcarbonylalkoxycarbonylthioalkyl” refers to a radical-alkyl-S—C(O)-alkoxy-C(O)—NH-alkyl or-alkyl-S—C(O)-alkoxy-C(O)-alkyl-NH₂, where alkyl and alkoxy are asdefined herein.

The term “alkoxycarbonylaminoalkyl” refers to a radical-alkyl-NH—C(O)-alkoxy or —NH-alkyl-C(O)-alkoxy, where alkyl and alkoxyare as defined herein.

The term “hydroxylalkyl” refers to a radical -alkyl-OH, where alkyl isas defined herein.

The term “aminoalkylcarbonylalkoxyl” refers to a radical-alkoxy-C(O)-alkyl-NH₂ or -alkoxy-C(O)—NH-alkyl, where alkyl and alkoxyare as defined herein.

“Amino” refers to the radical —NH₂ or —NH—R, where each R isindependently alkyl, aryl, or cycloalkyl.

“Amino alcohol” refers to the radical —NHLOH, wherein L is alkylene.

“Carboxyl” or “carboxy” refers to the radical —C(O)OH.

The term “alkylamino” or “arylamino” refers to an amino group that hasone or two alkyl or aryl substituents, respectively. In certainembodiments, the alkyl substituent is upper alkyl. In certainembodiments, the alkyl substituent is lower alkyl. In anotherembodiment, the alkyl, upper alkyl, or lower alkyl is unsubstituted.

“Halogen” or “halo” refers to chloro, bromo, fluoro or iodo.

“Monoalkylamino” refers to the group alkyl-NR′—, wherein R′ is selectedfrom hydrogen and alkyl or cycloalkyl.

“Thioalkoxy” refers to the group —SR′ where R′ is alkyl or cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a monovalentmonocyclic non-aromatic ring system and/or multicyclic ring system thatcontains at least one non-aromatic ring, wherein one or more of thenon-aromatic ring atoms are heteroatoms independently selected from O,S, or N; and the remaining ring atoms are carbon atoms. In certainembodiments, the heterocyclyl or heterocyclic group has from 3 to 20,from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6ring atoms. Heterocyclyl groups are bonded to the rest of the moleculethrough the non-aromatic ring. In certain embodiments, the heterocyclylis a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, whichmay include a fused or bridged ring system, and in which the nitrogen orsulfur atoms may be optionally oxidized, the nitrogen atoms may beoptionally quaternized, and some rings may be partially or fullysaturated, or aromatic. The heterocyclyl may be attached to the mainstructure at any heteroatom or carbon atom which results in the creationof a stable compound. Examples of such heterocyclic radicals include,but are not limited to, azepinyl, benzodioxanyl, benzodioxolyl,benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl,benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, β-carbolinyl,chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl,dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl,dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl,dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl,1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl,isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl,isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl,pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl,and 1,3,5-trithianyl. In certain embodiments, heterocyclic may also beoptionally substituted as described herein.

The term “heteroaryl” refers to refers to a monovalent monocyclicaromatic group and/or multicyclic aromatic group that contain at leastone aromatic ring, wherein at least one aromatic ring contains one ormore heteroatoms independently selected from O, S and N in the ring.Heteroaryl groups are bonded to the rest of the molecule through thearomatic ring. Each ring of a heteroaryl group can contain one or two Oatoms, one or two S atoms, and/or one to four N atoms, provided that thetotal number of heteroatoms in each ring is four or less and each ringcontains at least one carbon atom. In certain embodiments, theheteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms.Examples of monocyclic heteroaryl groups include, but are not limitedto, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl,oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl,triazinyl and triazolyl. Examples of bicyclic heteroaryl groups include,but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl,benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl,imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl,isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl,naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl,pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl,thiadiazolopyrimidyl, and thienopyridyl. Examples of tricyclicheteroaryl groups include, but are not limited to, acridinyl,benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl,phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyland xanthenyl. In certain embodiments, heteroaryl may also be optionallysubstituted as described herein.

The term “alkylaryl” refers to an aryl group with an alkyl substituent.The term “aralkyl” or “arylalkyl” refers to an alkyl group with an arylsubstituent.

The term “alkylheterocyclyl” refers to a heterocyclyl group with analkyl substituent. The term heterocyclylalkyl refers to an alkyl groupwith a heterocyclyl substituent.

The term “alkylheteroaryl” refers to a heteroaryl group with an alkylsubstituent. The term heteroarylalkyl refers to an alkyl group with aheteroaryl substituent.

The term “protecting group” as used herein and unless otherwise definedrefers to a group that is added to an oxygen, nitrogen or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis.

“Pharmaceutically acceptable salt” refers to any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art. Such salts include, but are not limited to: (1) acidaddition salts formed with organic or inorganic acids such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic,acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) baseaddition salts formed when an acidic proton present in the parentcompound either (a) is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion or an aluminum ion, or alkali metal oralkaline earth metal hydroxides, such as sodium, potassium, calcium,magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)coordinates with an organic base, such as aliphatic, alicyclic, oraromatic organic amines, such as ammonia, methylamine, dimethylamine,diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine,ethylenediamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, N-methylglucamine piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Pharmaceutically acceptable salts further include, by way of exampleonly and without limitation, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium and the like, and when the compoundcontains a basic functionality, salts of non-toxic organic or inorganicacids, such as hydrohalides, e.g. hydrochloride and hydrobromide,sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate,trichloroacetate, propionate, hexanoate, cyclopentylpropionate,glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate,ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate,3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate,laurate, methanesulfonate (mesylate), ethanesulfonate,1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate(besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate,4-toluenesulfonate, camphorate, camphorsulfonate,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate,gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate,cyclohexylsulfamate, quinate, muconate and the like.

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. In certain embodiments, a nucleobase is apurine or pyrimidine base, as defined herein.

The term “purine” or “pyrimidine” base refers to, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-iodopyrimidine, C⁶-iodo-pyrimidine, C⁵—Br-vinyl pyrimidine,C⁶—Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine,2,6-diaminopurine, and 6-chloropurine. Functional oxygen and nitrogengroups on the base can be protected as necessary or desired. Suitableprotecting groups are well known to those skilled in the art, andinclude trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such asacetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

The term “acyl” or “O-linked ester” refers to a group of the formulaC(O)R′, wherein R′ is alkyl or cycloalkyl (including lower alkyl),carboxylate reside of amino acid, aryl including phenyl, alkaryl,arylalkyl including benzyl, alkoxyalkyl including methoxymethyl,aryloxyalkyl such as phenoxymethyl; or substituted alkyl (includinglower alkyl), aryl including phenyl optionally substituted with chloro,bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy, sulfonate esterssuch as alkyl or arylalkyl sulphonyl including methanesulfonyl, themono, di or triphosphate ester, trityl or monomethoxy-trityl,substituted benzyl, alkaryl, arylalkyl including benzyl, alkoxyalkylincluding methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groupsin the esters optimally comprise a phenyl group. In particular, acylgroups include acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl,propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoroheptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nonafluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxybenzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

The term “amino acid” refers to naturally occurring and synthetic α, β γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In certain embodiments, the amino acid is in theL-configuration. Alternatively, the amino acid can be a derivative ofalanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl.

The term “amino acid derivative” refers to a group derivable from anaturally or non-naturally occurring amino acid, as described andexemplified herein. Amino acid derivatives are apparent to those ofskill in the art and include, but are not limited to, ester, aminoalcohol, amino aldehyde, amino lactone, and N-methyl derivatives ofnaturally and non-naturally occurring amino acids. In an embodiment, anamino acid derivative is provided as a substituent of a compounddescribed herein, wherein the substituent is —NH-G(S_(C))—C(O)-Q or—OC(O)G(S_(C))-Q, wherein Q is —SR, —NRR or alkoxyl, R is hydrogen oralkyl, S_(C) is a side chain of a naturally occurring or non-naturallyoccurring amino acid and G is C₁-C₂ alkyl. In certain embodiments, G isC₁ alkyl and S_(C) is selected from the group consisting of hydrogen,alkyl, heteroalkyl, arylalkyl and heteroarylalkyl.

As used herein, the term “hydantoinyl” refers to the group

where R^(XX) and R^(YY) are each independently hydrogen or lower alkyl.

As used herein, the term “hydantoinylalkyl” refers to the group-alkyl-hydantoinyl, where alkyl and hydantoinyl are as described herein.

The term “substantially free of” or “substantially in the absence of”with respect to a nucleoside composition refers to a nucleosidecomposition that includes at least 85 or 90% by weight, in certainembodiments 95%, 98%, 99% or 100% by weight, of the designatedenantiomer of that nucleoside. In certain embodiments, in the methodsand compounds provided herein, the compounds are substantially free ofenantiomers.

Similarly, the term “isolated” with respect to a nucleoside compositionrefers to a nucleoside composition that includes at least 85, 90%, 95%,98%, 99% to 100% by weight, of the nucleoside, the remainder comprisingother chemical species or enantiomers.

“Solvate” refers to a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

“Isotopic composition” refers to the amount of each isotope present fora given atom, and “natural isotopic composition” refers to the naturallyoccurring isotopic composition or abundance for a given atom. Atomscontaining their natural isotopic composition may also be referred toherein as “non-enriched” atoms. Unless otherwise designated, the atomsof the compounds recited herein are meant to represent any stableisotope of that atom. For example, unless otherwise stated, when aposition is designated specifically as “H” or “hydrogen,” the positionis understood to have hydrogen at its natural isotopic composition.

“Isotopic enrichment” refers to the percentage of incorporation of anamount of a specific isotope at a given atom in a molecule in the placeof that atom's natural isotopic abundance. For example, deuteriumenrichment of 1% at a given position means that 1% of the molecules in agiven sample contain deuterium at the specified position. Because thenaturally occurring distribution of deuterium is about 0.0156%,deuterium enrichment at any position in a compound synthesized usingnon-enriched starting materials is about 0.0156%. The isotopicenrichment of the compounds provided herein can be determined usingconventional analytical methods known to one of ordinary skill in theart, including mass spectrometry and nuclear magnetic resonancespectroscopy.

“Isotopically enriched” refers to an atom having an isotopic compositionother than the natural isotopic composition of that atom. “Isotopicallyenriched” may also refer to a compound containing at least one atomhaving an isotopic composition other than the natural isotopiccomposition of that atom.

As used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,”“alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise deuterium at one or more positions where hydrogen atoms arepresent, and wherein the deuterium composition of the atom or atoms isother than the natural isotopic composition.

Also as used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “carboxyl,” “alkylamino,”“arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise carbon-13 at an amount other than the natural isotopiccomposition.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse in an assay that measures such response.

The term “host,” as used herein, refers to any unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and in certain embodiments, a human. Alternatively,the host can be carrying a part of the Flaviviridae viral genome, whosereplication or function can be altered by the compounds of the presentinvention. The term host specifically includes infected cells, cellstransfected with all or part of the Flaviviridae genome and animals, inparticular, primates (including chimpanzees) and humans. In most animalapplications of the present invention, the host is a human patient.Veterinary applications, in certain indications, however, are clearlyanticipated by the present invention (such as chimpanzees).

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), and for example, a human.In certain embodiments, the subject is refractory or non-responsive tocurrent treatments for hepatitis C infection. In another embodiment, thesubject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet(e.g., a dog or a cat). In certain embodiments, the subject is a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” includes a compound provided herein. Incertain embodiments, a therapeutic agent is an agent which is known tobe useful for, or has been or is currently being used for the treatmentor prevention of a disorder or one or more symptoms thereof.

“Therapeutically effective amount” refers to an amount of a compound orcomposition that, when administered to a subject for treating a disease,is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in certainembodiments, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” includesameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”includes modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” includes delaying the onset of thedisease or disorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” includes a compound provided herein. Incertain other embodiments, the term “prophylactic agent” does not refera compound provided herein. For example, a prophylactic agent is anagent which is known to be useful for, or has been or is currently beingused to prevent or impede the onset, development, progression and/orseverity of a disorder.

As used herein, the phrase “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention or reduction of the development, recurrenceor onset of one or more symptoms associated with a disorder, or toenhance or improve the prophylactic effect(s) of another therapy (e.g.,another prophylactic agent).

Compounds

Provided herein are D-amino acid compounds useful for the treatment ofliver diseases and conditions, for example, Flaviviridae infections suchas HCV infection. The D-amino acid compounds can be formed as describedherein and used for the treatment of, for example, Flaviviridaeinfections such as HCV infection.

In certain embodiments, provided herein are compounds according toformula (2001):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof, wherein: Base is anucleobase; A is S or O; W is S or O; X is a D-amino acid residue, or anester thereof; Y is hydrogen, —OR¹, —SR¹, or —NR¹R²; R^(b1) is alkyl,cycloalkyl, —H, azido, cyano, or halogen; R^(b2) is —OH, —Cl, —F, —H,azido, cyano, amino, or alkoxyl, or, in the alternative, R^(b1) andR^(b2), along with the carbon atom to which they are attached, join toform a three-membered carbocyclic or heterocyclic ring; R^(c) is —H or—OH, or, in the alternative, Y and R^(c) join to form a six-memberedheterocyclic ring wherein Y and R^(c) together represent a singledivalent —O—; R^(d) is —H, —F, azido, or allenyl; or, in thealternative, R^(b2) and R^(d) join to form alkylene or substitutedalkylene; R^(e) is —H or alkyl; each R¹ is independently alkyl,cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, substitutedalkyl or hydantoinylalkyl; and each R² is independently hydrogen oralkyl. In certain embodiments of Formula (2001), R^(b1) and R^(b2),along with the carbon atom to which they are attached, join to form athree-membered carbocyclic or heterocyclic ring. In certain embodiments,R^(b1) and R^(b2), along with the carbon atom to which they areattached, join to form cyclopropylene or oxiranylene.

In certain embodiments, provided herein are compounds according toFormula (I):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according toFormula (Ia) or (Ib):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof. In certain embodiments,R_(P) compounds are provided. In certain embodiments, S_(P) compoundsare provided.

In Formula (I), (Ia) or (Ib), Base is any nucleobase known to those ofskill in the art. Base can be a naturally occurring nucleobase, or itcan be a non-natural nucleobase known to those of skill in the art. Incertain embodiments, Base is a purine or pyrimidine nucleobase. Inparticular embodiments, Base is guanosine, uracil, cytosine, adenine ora derivative thereof. Exemplary nucleobases are described herein.

In Formula (I), (Ia) or (Ib), W is S or O. In certain embodiments, W isS. In certain embodiments, W is O.

In Formula (I), (Ia) or (Ib), X is a D-amino acid residue, or an esterthereof. X can be any D-amino acid residue known to those of skill inthe art. X can be the D-enantiomer of a naturally occurring amino acidresidue, or X can be the D-enantiomer of a non-natural amino acidresidue. In particular embodiments, X is D-alanine, D-phenylalanine,D-valine or D-terleucine. In preferred embodiments, X is D-alanine. Theester can be any ester known to those of skill in the art. In particularembodiments, the ester is an alkyl ester. In certain embodiments, theester is selected from the group consisting of ethyl ester, propylester, n-propyl ester, isopropyl ester, butyl ester, t-butyl ester,n-butyl ester, and cyclopentyl ester.

In Formula (I), (Ia) or (Ib), Y is hydrogen, —OR¹, —SR¹, or —NR¹R². EachR¹ is independently alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, substituted alkyl or hydantoinylalkyl. In certainembodiments, each R¹ is independently alkyl, cycloalkyl,heterocyclylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,alkylcarbonylthioalkyl, alkoxycarbonylalkyl, arylalkoxycarbonylalkyl,alkylcarbonylalkoxy(arylalkyl),(alkoxycarbonyl)(alkoxycarbonylamino)alkyl, cycloalkylcarbonylalkoxyl,alkoxycarbonylaminoalkylcarbonylthioalkyl,hydroxylalkylcarbonylthioalkyl,aminoalkylcarbonylalkoxycarbonylthioalkyl, or hydantoinylalkyl. Each R²is independently hydrogen or alkyl. In particular embodiments, R² is H.

In Formula (I), (Ia) or (Ib), R^(c) is —H or —OH. In the alternative, incertain embodiments, Y and R^(c) join to form a six-memberedheterocyclic ring wherein Y and R^(c) together represent a singledivalent —O—. In these embodiments, the compounds comprise a cyclicphosphate group linking the 3′ and 5′ carbons of the nucleoside sugar.

In Formula (I), (Ia) or (Ib), R^(b1) is —CH₃, —H, azido, cyano, orhalogen. In certain embodiments, R^(b1) is —CH₃. Also in Formula (I),(Ia) or (Ib), R^(b2) is —OH, —Cl, —F, —H, azido, cyano, amino, oralkoxyl. In certain embodiments, R^(b2) is —OH. In certain embodiments,R^(b2) is —Cl. In certain embodiments, R^(b2) is —F. In certainembodiments of Formula (I), (Ia) or (Ib), R^(b1) and R^(b2), along withthe carbon atom to which they are attached, join to form athree-membered carbocyclic or heterocyclic ring. In certain embodiments,R^(b1) and R^(b2), along with the carbon atom to which they areattached, join to form cyclopropylene or oxiranylene.

In Formula (I), (Ia) or (Ib), R^(d) is —H, —F, azido, or allenyl. Incertain embodiments, R^(d) is —H. In the alternative, in certainembodiments, R^(b2) and R^(d) join to form alkylene or substitutedalkylene. In particular embodiments, R^(b2) and R^(d) form —CH₂—O—. Inparticular embodiments, the —CH₂— is linked to the 4′ carbon of thesugar, and the —O— is linked to the 2′ carbon of the sugar.

In Formula (I), (Ia) or (Ib), R^(e) is —H or alkyl. In particularembodiments, R^(e) is —H.

In certain embodiments according to Formula (I), (Ia) or (Ib), R^(e) is—H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is —Hor —OH; and R² is H. In particular embodiments, R^(e) is —H; R^(d) is—H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is —H or —OH; R² isH; and Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof. In particular embodiments, R^(e) is —H; R^(d) is —H;R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is —H or —OH; R² is H;Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof; and X is D-alanine, or an ester thereof. In certainembodiments according to this paragraph, Y is alkyl, aryl, arylalkyl,cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl.

In certain embodiments according to Formula (I), (Ia) or (Ib), R^(e) is—H; R^(b2) and R^(d) form —CH₂—O—; R^(b1) is —CH₃; R^(c) is —H or —OH;and R² is H. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂—O—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; and Base isselected from guanosine, uracil, cytosine, adenine or a derivativethereof. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂—O—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; Base is selectedfrom guanosine, uracil, cytosine, adenine or a derivative thereof; and Xis D-alanine, or an ester thereof. In certain embodiments according tothis paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl.

In certain embodiments according to Formula (I), (Ia) or (Ib), R^(e) is—H; R^(b2) and R^(d) form —CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or —OH;and R² is H. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; and Base isselected from guanosine, uracil, cytosine, adenine or a derivativethereof. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; Base is selectedfrom guanosine, uracil, cytosine, adenine or a derivative thereof; and Xis D-alanine, or an ester thereof. In certain embodiments according tothis paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl.

In certain embodiments according to Formula (I), (Ia) or (Ib), R^(e) is—H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R² is H; andY and R^(c) together represent a single divalent —O—. In particularembodiments, R^(e) is —H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH,—Cl or —F; R² is H; Y and R^(c) together represent a single divalent—O—; and Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof. In particular embodiments, R^(e) is —H; R^(d) is —H;R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R² is H; Y and R^(c) togetherrepresent a single divalent —O—; and Base is selected from guanosine,uracil, cytosine, adenine or a derivative thereof; and X is D-alanine,or an ester thereof. In certain embodiments according to this paragraph,Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl.

In certain embodiments, provided herein are compounds according toFormula (II):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according toFormula (IIa) or (IIb):

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof. In certain embodiments,R_(P) compounds are provided. In certain embodiments, S_(P) compoundsare provided.

In Formulas (II), (IIa) and (IIb), the symbols R^(b1), R^(b2), R^(c),R^(d), R^(e), W, Y and Base have the meanings provided above.

In Formulas (II), (IIa) and (IIb), each R¹⁰ is independently alkyl,arylalkyl, heteroarylalkyl or a side chain of a naturally occurringamino acid, other than hydrogen. In particular embodiments, R¹⁰ ismethyl, isopropyl, t-butyl or benzyl.

In Formulas (II), (IIa) and (IIb), each R¹¹ is independently alkyl,cycloalkyl or —H. In particular embodiments, each R¹¹ is ethyl, propyl,isopropyl, n-propyl, butyl, n-butyl, t-butyl or cyclopentyl.

In certain embodiments of Formulas (II), (IIa) and (IIb), R^(c) is —H or—OH. In the alternative, in certain embodiments, Y and R^(c) join toform a six-membered heterocyclic ring wherein Y and R^(c) togetherrepresent a single divalent —O—. In these embodiments, the compoundscomprise a cyclic phosphate group linking the 3′ and 5′ carbons of thenucleoside sugar.

In certain embodiments of Formulas (II), (IIa) and (IIb), R^(b1) is—CH₃. Also in certain embodiments, R^(b2) is —OH, —Cl or —F. In certainembodiments, R^(b2) is —OH. In certain embodiments, R^(b2) is —Cl. Incertain embodiments, R^(b2) is —F.

In certain embodiments of Formulas (II), (IIa) and (IIb), R^(d) is —H.In the alternative, in certain embodiments, R^(b2) and R^(d) join toform alkylene or substituted alkylene. In particular embodiments, R^(b2)and R^(d) form —CH₂—O—. In particular embodiments, the —CH₂— is linkedto the 4′ carbon of the sugar, and the —O— is linked to the 2′ carbon ofthe sugar.

In certain embodiments of Formulas (II), (IIa) and (IIb), R^(e) is —H oralkyl. In particular embodiments, R^(e) is —H.

In certain embodiments according to Formula (II), (IIa) or (IIb), R^(e)is —H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is—H or —OH; and R² is H. In particular embodiments, R^(e) is —H; R^(d) is—H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is —H or —OH; R² isH; and Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof. In particular embodiments, R^(e) is —H; R^(d) is —H;R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R^(c) is —H or —OH; R² is H;Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof; and X is D-alanine, or an ester thereof. In certainembodiments according to this paragraph, Y is alkyl, aryl, arylalkyl,cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl. In particular embodiments according to thisparagraph, R¹⁰ is methyl, isopropyl, t-butyl or benzyl; and R¹¹ isethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl orcyclopentyl.

In certain embodiments according to Formula (II), (IIa) or (IIb), R^(e)is —H; R^(b2) and R^(d) form —CH₂—O—; R^(b1) is —CH₃; R^(e) is —H or—OH; and R² is H. In certain embodiments, R^(e) is —H; R^(b2) and R^(d)form —CH₂—O—; R^(b1) is —CH₃; R^(e) is —H or —OH; R² is H; and Base isselected from guanosine, uracil, cytosine, adenine or a derivativethereof. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂—O—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; Base is selectedfrom guanosine, uracil, cytosine, adenine or a derivative thereof; and Xis D-alanine, or an ester thereof. In certain embodiments according tothis paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl. In particular embodiments according to thisparagraph, R¹⁰ is methyl, isopropyl, t-butyl or benzyl; and R¹¹ isethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl orcyclopentyl.

In certain embodiments according to Formula (II), (IIa) or (IIb), R^(e)is —H; R^(b2) and R^(d) form —CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or—OH; and R² is H. In certain embodiments, R^(e) is —H; R^(b2) and R^(d)form —CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; and Base isselected from guanosine, uracil, cytosine, adenine or a derivativethereof. In certain embodiments, R^(e) is —H; R^(b2) and R^(d) form—CH₂CH₂—; R^(b1) is —CH₃; R^(c) is —H or —OH; R² is H; Base is selectedfrom guanosine, uracil, cytosine, adenine or a derivative thereof; and Xis D-alanine, or an ester thereof. In certain embodiments according tothis paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl. In particular embodiments according to thisparagraph, R¹⁰ is methyl, isopropyl, t-butyl or benzyl; and R¹¹ isethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl orcyclopentyl.

In certain embodiments according to Formula (II), (IIa) or (IIb), R^(e)is —H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R² is H;and Y and R^(c) together represent a single divalent —O—. In particularembodiments, R^(e) is —H; R^(d) is —H; R^(b1) is —CH₃; R^(b2) is —OH,—Cl or —F; R² is H; Y and R^(c) together represent a single divalent—O—; and Base is selected from guanosine, uracil, cytosine, adenine or aderivative thereof. In particular embodiments, R^(e) is —H; R^(d) is —H;R^(b1) is —CH₃; R^(b2) is —OH, —Cl or —F; R² is H; Y and R^(c) togetherrepresent a single divalent —O—; and Base is selected from guanosine,uracil, cytosine, adenine or a derivative thereof; and X is D-alanine,or an ester thereof. In certain embodiments according to this paragraph,Y is alkyl, aryl, arylalkyl, cycloalkyl or

wherein R³ is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, oraminoalkylcarbonylalkoxyl. In particular embodiments according to thisparagraph, R¹⁰ is methyl, isopropyl, t-butyl or benzyl; and R¹¹ isethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl orcyclopentyl.

In certain embodiments, a compound of any of Formulas (I), (Ia), (Ib),(II), (IIa), or (IIb) is provided wherein: each Base is independently

or a tautomer thereof; each R⁴ is independently hydrogen, hydroxyl,hydroxylamine, alkylamino, halogen, sulfanyl, amino or alkoxy; each R⁵is independently hydrogen, halogen or methyl; and each R⁶ isindependently hydrogen, amino, or halo.

In certain embodiments, a compound of any of Formulas (I), (Ia), (Ib),(II), (IIa) or (IIb) is provided wherein: each Base is independently

or a tautomer thereof; each R⁴ is independently hydrogen, hydroxyl,hydroxylamine, halogen, sulfanyl, amino or alkoxy; and each R⁵ isindependently hydrogen, halogen or methyl. In an embodiment, each R⁴ isalkylamino. In an embodiment, each R⁴ is alkylamino having from seven tothirty carbon atoms. In an embodiment, each R⁴ is alkylamino having fromfifteen to thirty carbon atoms. In an embodiment, each R⁴ is alkylaminohaving from twenty to thirty carbon atoms. In an embodiment, each R⁴ isalkylamino having from seven to fifteen carbon atoms. In an embodiment,each R⁴ is alkylamino having from seven to twenty carbon atoms. In anembodiment, each R⁴ is alkylamino having from ten to twenty carbonatoms.

In certain embodiments, a compound of any of the following Formulas isprovided:

or pharmaceutically acceptable salts, solvates, stereoisomeric forms orpolymorphic forms thereof, wherein: R^(b1), R^(b2), R^(c), R^(d), R^(e),W, X, and Y are as defined in the context of Formula (I); and each R⁵ isindependently hydrogen, halogen or methyl.

In certain embodiments, provided herein are compounds according to anyof the following Formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof, wherein R^(b1), R^(b2),R^(c), R^(d), R^(e), W, X, and Y are as defined in the context ofFormula (I). In certain embodiments, a compound of Formula (XVII) isprovided. In certain embodiments, a compound of Formula (XVIII) isprovided. In certain embodiments, a compound of Formula (XIX) isprovided. In certain embodiments, a compound of Formula (XX) isprovided.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, a compound of formula according to formula 401or 425 is provided:

or a pharmaceutically acceptable salt, solvate, phosphate, prodrug,stereoisomeric form, tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments provided herein are compounds according to any ofthe following formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In certain embodiments, provided herein are compounds according to anyof the following Formulas:

or a pharmaceutically acceptable salt, solvate, stereoisomeric form,tautomeric form or polymorphic form thereof.

In an embodiment, provided herein are compounds comprising a D-aminoacid, or ester thereof, linked to a drug. In certain embodiments, thedrug is a drug for treating a liver disease or condition. In certainembodiments, the liver disease or condition is hepatitis, fatty liverdisease, cirrhosis, liver cancer, biliary cirrhosis, sclerosingcholangitis, Budd-Chiari syndrome, hemochromatosis, Wilson's disease,Gilbert's syndrome, biliary atresia, alpha-1 antitrypsin deficiency,alagille syndrome, or progressive familial intrahepatic cholestasis. Incertain embodiments, the drug is a drug for treating hepatitis C. Incertain embodiments, the drug is an interferon, a nucleotide analogue, apolymerase inhibitor, an NS3 protease inhibitor, an NS5A inhibitor, anentry inhibitor, a non-nucleoside polymerase inhibitor, a cyclosporineimmune inhibitor, an NS4A antagonist, an NS4B-RNA binding inhibitor, alocked nucleic acid mRNA inhibitor, or a cyclophilin inhibitor.

In some embodiments, provided herein are:

-   (a) compounds as described herein and pharmaceutically acceptable    salts and compositions thereof;-   (b) compounds as described herein and pharmaceutically acceptable    salts and compositions thereof for use in the treatment and/or    prophylaxis of a liver disorder including Flaviviridae infection,    especially in individuals diagnosed as having a Flaviviridae    infection or being at risk of becoming infected by hepatitis C;-   (c) processes for the preparation of compounds as described herein    as described in more detail elsewhere herein;-   (d) pharmaceutical formulations comprising a compound as described    herein, or a pharmaceutically acceptable salt thereof together with    a pharmaceutically acceptable carrier or diluent;-   (e) pharmaceutical formulations comprising a compound as described    herein or a pharmaceutically acceptable salt thereof together with    one or more other effective anti-HCV agents, optionally in a    pharmaceutically acceptable carrier or diluent;-   (f) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a compound as described herein its pharmaceutically    acceptable salt or composition; or-   (g) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a compounds as described herein, its pharmaceutically    acceptable salt or composition in combination and/or alternation    with one or more effective anti-HCV agent.

Optically Active Compounds

It is appreciated that compounds provided herein have several chiralcenters and may exist in and be isolated in optically active and racemicforms. Some compounds may exhibit polymorphism. It is to be understoodthat any racemic, optically-active, diastereomeric, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound provided herein,which possess the useful properties described herein is within the scopeof the invention. It being well known in the art how to prepareoptically active forms (for example, by resolution of the racemic formby recrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

In particular, since the 1′ and 4′ carbons of a nucleoside are chiral,their non-hydrogen substituents (the base and the CHOR groups,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the sugar ring system. The four optical isomerstherefore are represented by the following configurations (whenorienting the sugar moiety in a horizontal plane such that the oxygenatom is in the back): cis (with both groups “up”, which corresponds tothe configuration of naturally occurring β-D nucleosides), cis (withboth groups “down”, which is a non-naturally occurring β-Lconfiguration), trans (with the C2′ substituent “up” and the C4′substituent “down”), and trans (with the C2′ substituent “down” and theC4′ substituent “up”). The “D-nucleosides” are cis nucleosides in anatural configuration and the “L-nucleosides” are cis nucleosides in thenon-naturally occurring configuration.

Likewise, most amino acids are chiral (designated as L or D, wherein theL enantiomer is the naturally occurring configuration) and can exist asseparate enantiomers.

Examples of methods to obtain optically active materials are known inthe art, and include at least the following.

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

In some embodiments, compositions of 2′-chloro nucleoside analogcompounds that are substantially free of a designated enantiomer of thatcompound. In certain embodiments, in the methods and compounds of thisinvention, the compounds are substantially free of enantiomers. In someembodiments, the composition includes that includes a compound that isat least 85, 90%, 95%, 98%, 99% to 100% by weight, of the compound, theremainder comprising other chemical species or enantiomers.

Isotopically Enriched Compounds

Also provided herein are isotopically enriched compounds, including butnot limited to isotopically enriched 2′-chloro nucleoside analogcompounds.

Isotopic enrichment (for example, deuteration) of pharmaceuticals toimprove pharmacokinetics (“PK”), pharmacodynamics (“PD”), and toxicityprofiles, has been demonstrated previously with some classes of drugs.See, for example, Lijinsky et. al., Food Cosmet. Toxicol., 20: 393(1982); Lijinsky et. al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangoldet. al., Mutation Res. 308: 33 (1994); Gordon et. al., Drug Metab.Dispos., 15: 589 (1987); Zello et. al., Metabolism, 43: 487 (1994);Gately et. al., J. Nucl. Med., 27: 388 (1986); Wade D, Chem. Biol.Interact. 117: 191 (1999).

Isotopic enrichment of a drug can be used, for example, to (1) reduce oreliminate unwanted metabolites, (2) increase the half-life of the parentdrug, (3) decrease the number of doses needed to achieve a desiredeffect, (4) decrease the amount of a dose necessary to achieve a desiredeffect, (5) increase the formation of active metabolites, if any areformed, and/or (6) decrees the production of deleterious metabolites inspecific tissues and/or create a more effective drug and/or a safer drugfor combination therapy, whether the combination therapy is intentionalor not.

Replacement of an atom for one of its isotopes often will result in achange in the reaction rate of a chemical reaction. This phenomenon isknown as the Kinetic Isotope Effect (“KIE”). For example, if a C—H bondis broken during a rate-determining step in a chemical reaction (i.e.the step with the highest transition state energy), substitution of adeuterium for that hydrogen will cause a decrease in the reaction rateand the process will slow down. This phenomenon is known as theDeuterium Kinetic Isotope Effect (“DKIE”). (See, e.g., Foster et al.,Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J.Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).

The magnitude of the DKIE can be expressed as the ratio between therates of a given reaction in which a C—H bond is broken, and the samereaction where deuterium is substituted for hydrogen. The DKIE can rangefrom about 1 (no isotope effect) to very large numbers, such as 50 ormore, meaning that the reaction can be fifty, or more, times slower whendeuterium is substituted for hydrogen. High DKIE values may be due inpart to a phenomenon known as tunneling, which is a consequence of theuncertainty principle. Tunneling is ascribed to the small mass of ahydrogen atom, and occurs because transition states involving a protoncan sometimes form in the absence of the required activation energy.Because deuterium has more mass than hydrogen, it statistically has amuch lower probability of undergoing this phenomenon.

Tritium (“T”) is a radioactive isotope of hydrogen, used in research,fusion reactors, neutron generators and radiopharmaceuticals. Tritium isa hydrogen atom that has 2 neutrons in the nucleus and has an atomicweight close to 3. It occurs naturally in the environment in very lowconcentrations, most commonly found as T₂O. Tritium decays slowly(half-life=12.3 years) and emits a low energy beta particle that cannotpenetrate the outer layer of human skin. Internal exposure is the mainhazard associated with this isotope, yet it must be ingested in largeamounts to pose a significant health risk. As compared with deuterium, alesser amount of tritium must be consumed before it reaches a hazardouslevel. Substitution of tritium (“T”) for hydrogen results in yet astronger bond than deuterium and gives numerically larger isotopeeffects. Similarly, substitution of isotopes for other elements,including, but not limited to, ¹³C or ¹⁴C for carbon, ³³S, ³⁴S, or ³⁶Sfor sulfur, ¹⁵N for nitrogen, and ¹⁷O or ¹⁸O for oxygen, may lead to asimilar kinetic isotope effect.

For example, the DKIE was used to decrease the hepatotoxicity ofhalothane by presumably limiting the production of reactive species suchas trifluoroacetyl chloride. However, this method may not be applicableto all drug classes. For example, deuterium incorporation can lead tometabolic switching. The concept of metabolic switching asserts thatxenogens, when sequestered by Phase I enzymes, may bind transiently andre-bind in a variety of conformations prior to the chemical reaction(e.g., oxidation). This hypothesis is supported by the relatively vastsize of binding pockets in many Phase I enzymes and the promiscuousnature of many metabolic reactions. Metabolic switching can potentiallylead to different proportions of known metabolites as well as altogethernew metabolites. This new metabolic profile may impart more or lesstoxicity.

The animal body expresses a variety of enzymes for the purpose ofeliminating foreign substances, such as therapeutic agents, from itscirculation system. Examples of such enzymes include the cytochrome P450enzymes (“CYPs”), esterases, proteases, reductases, dehydrogenases, andmonoamine oxidases, to react with and convert these foreign substancesto more polar intermediates or metabolites for renal excretion. Some ofthe most common metabolic reactions of pharmaceutical compounds involvethe oxidation of a carbon-hydrogen (C—H) bond to either a carbon-oxygen(C—O) or carbon-carbon (C—C) pi-bond. The resultant metabolites may bestable or unstable under physiological conditions, and can havesubstantially different pharmacokinetic, pharmacodynamic, and acute andlong-term toxicity profiles relative to the parent compounds. For manydrugs, such oxidations are rapid. These drugs therefore often requirethe administration of multiple or high daily doses.

Therefore, isotopic enrichment at certain positions of a compoundprovided herein will produce a detectable KIE that will affect thepharmacokinetic, pharmacologic, and/or toxicological profiles of acompound provided herein in comparison with a similar compound having anatural isotopic composition.

Preparation of Compounds

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Compounds providedherein can be prepared according to the Exemplary Preparation Schemes inthe Examples provided below. Reaction conditions, steps and reactantsnot provided in the Exemplary Preparation Schemes would be apparent to,and known by, those skilled in the art.

Pharmaceutical Compositions and Methods of Administration

Compounds provided herein can be formulated into pharmaceuticalcompositions using methods available in the art and those disclosedherein. Any of the compounds disclosed herein can be provided in theappropriate pharmaceutical composition and be administered by a suitableroute of administration.

The methods provided herein encompass administering pharmaceuticalcompositions containing at least one compound as described herein, ifappropriate in the salt form, either used alone or in the form of acombination with one or more compatible and pharmaceutically acceptablecarriers, such as diluents or adjuvants, or with another anti-HCV agent.

In certain embodiments, the second agent can be formulated or packagedwith the compound provided herein. Of course, the second agent will onlybe formulated with the compound provided herein when, according to thejudgment of those of skill in the art, such co-formulation should notinterfere with the activity of either agent or the method ofadministration. In certain embodiments, the compound provided herein andthe second agent are formulated separately. They can be packagedtogether, or packaged separately, for the convenience of thepractitioner of skill in the art.

In clinical practice the active agents provided herein may beadministered by any conventional route, in particular orally,parenterally, rectally or by inhalation (e.g. in the form of aerosols).In certain embodiments, the compound provided herein is administeredorally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product is mixed with one or more inertdiluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release.

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, for example wetting, sweetening or flavoring products.

The compositions for parenteral administration can be emulsions orsterile solutions. Use may be made, as solvent or vehicle, of propyleneglycol, a polyethylene glycol, vegetable oils, in particular olive oil,or injectable organic esters, for example ethyl oleate. Thesecompositions can also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be carried out in several ways, for example using abacteriological filter, by radiation or by heating. They can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active principle, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, for example dextran, mannitol or lactose.

In certain embodiments, a composition provided herein is apharmaceutical composition or a single unit dosage form. Pharmaceuticalcompositions and single unit dosage forms provided herein comprise aprophylactically or therapeutically effective amount of one or moreprophylactic or therapeutic agents (e.g., a compound provided herein, orother prophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In a specificembodiment and in this context, the term “pharmaceutically acceptable”means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” includes a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water can be used as a carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

Further encompassed herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, New York, 1995, pp. 379 80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine can be anhydrousif substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

Further provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent, in certain embodiments, in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject. The formulation shouldsuit the mode of administration. In a certain embodiment, thepharmaceutical compositions or single unit dosage forms are sterile andin suitable form for administration to a subject, for example, an animalsubject, such as a mammalian subject, for example, a human subject.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal,sublingual, inhalation, intranasal, transdermal, topical, transmucosal,intra-tumoral, intra-synovial and rectal administration. In a specificembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenous,subcutaneous, intramuscular, oral, intranasal or topical administrationto human beings. In an embodiment, a pharmaceutical composition isformulated in accordance with routine procedures for subcutaneousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocamne to ease pain at the site of theinjection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a subject,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil in water emulsions, or a water in oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a subject; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a subject.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of viral infection may contain larger amountsof one or more of the active ingredients it comprises than a dosage formused in the maintenance treatment of the same infection. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed herein will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms comprise a compound provided herein, or apharmaceutically acceptable salt, solvate or hydrate thereof lie withinthe range of from about 0.1 mg to about 1000 mg per day, given as asingle once-a-day dose in the morning or as divided doses throughout theday taken with food. Particular dosage forms can have about 0.1, 0.2,0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100,200, 250, 500 or 1000 mg of the active compound.

Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

In certain embodiments, the oral dosage forms are solid and preparedunder anhydrous conditions with anhydrous ingredients, as described indetail herein. However, the scope of the compositions provided hereinextends beyond anhydrous, solid oral dosage forms. As such, furtherforms are described herein.

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or non-aqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms include,but are not limited to, binders, fillers, disintegrants, and lubricants.Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch,hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is typically presentin from about 50 to about 99 weight percent of the pharmaceuticalcomposition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC581, AVICEL PH 105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Aspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL PH 103™ and Starch 1500LM.

Disintegrants are used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients should be used to form solid oral dosage forms.The amount of disintegrant used varies based upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. Typical pharmaceutical compositions comprise from about 0.5 toabout 15 weight percent of disintegrant, specifically from about 1 toabout 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, pre gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulatedaerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.),CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. ofBoston, Mass.), and mixtures thereof. If used at all, lubricants aretypically used in an amount of less than about 1 weight percent of thepharmaceutical compositions or dosage forms into which they areincorporated.

Delayed Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and6,699,500; each of which is incorporated herein by reference in itsentirety. Such dosage forms can be used to provide slow or controlledrelease of one or more active ingredients using, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled releaseformulations known to those of ordinary skill in the art, includingthose described herein, can be readily selected for use with the activeingredients provided herein. Thus encompassed herein are single unitdosage forms suitable for oral administration such as, but not limitedto, tablets, capsules, gelcaps, and caplets that are adapted forcontrolled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In certain embodiments, a pump may beused (see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed in asubject at an appropriate site determined by a practitioner of skill,i.e., thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138(1984)). Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)). The active ingredient can bedispersed in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

Parenteral Dosage Forms

In certain embodiments, provided are parenteral dosage forms. Parenteraldosage forms can be administered to subjects by various routesincluding, but not limited to, subcutaneous, intravenous (includingbolus injection), intramuscular, and intraarterial. Because theiradministration typically bypasses subjects' natural defenses againstcontaminants, parenteral dosage forms are typically, sterile or capableof being sterilized prior to administration to a subject. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms.

Transdermal, Topical & Mucosal Dosage Forms

Also provided are transdermal, topical, and mucosal dosage forms.Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 16^(th), 18th and 20^(th) eds., Mack Publishing, Easton Pa.(1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms,4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels. Further, transdermal dosage forms include“reservoir type” or “matrix type” patches, which can be applied to theskin and worn for a specific period of time to permit the penetration ofa desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed herein are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are nontoxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18th and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 & 2000).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients provided. For example, penetration enhancers canbe used to assist in delivering the active ingredients to the tissue.Suitable penetration enhancers include, but are not limited to: acetone;various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkylsulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethylformamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the infection andother factors specific to the subject to be treated. In certainembodiments, doses are from about 1 to about 1000 mg per day for anadult, or from about 5 to about 250 mg per day or from about 10 to 50 mgper day for an adult. In certain embodiments, doses are from about 5 toabout 400 mg per day or 25 to 200 mg per day per adult. In certainembodiments, dose rates of from about 50 to about 500 mg per day arealso contemplated.

In further aspects, provided are methods of treating or preventing anHCV infection in a subject by administering, to a subject in needthereof, an effective amount of a compound provided herein, or apharmaceutically acceptable salt thereof. The amount of the compound orcomposition which will be effective in the prevention or treatment of adisorder or one or more symptoms thereof will vary with the nature andseverity of the disease or condition, and the route by which the activeingredient is administered. The frequency and dosage will also varyaccording to factors specific for each subject depending on the specifictherapy (e.g., therapeutic or prophylactic agents) administered, theseverity of the disorder, disease, or condition, the route ofadministration, as well as age, body, weight, response, and the pastmedical history of the subject. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

In certain embodiments, exemplary doses of a composition includemilligram or microgram amounts of the active compound per kilogram ofsubject or sample weight (e.g., about 10 micrograms per kilogram toabout 50 milligrams per kilogram, about 100 micrograms per kilogram toabout 25 milligrams per kilogram, or about 100 microgram per kilogram toabout 10 milligrams per kilogram). For compositions provided herein, incertain embodiments, the dosage administered to a subject is 0.140 mg/kgto 3 mg/kg of the subject's body weight, based on weight of the activecompound. In certain embodiments, the dosage administered to a subjectis between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50mg/kg of the subject's body weight.

In certain embodiments, the recommended daily dose range of acomposition provided herein for the conditions described herein liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose or as divided doses throughout a day. Incertain embodiments, the daily dose is administered twice daily inequally divided doses. In certain embodiments, a daily dose range shouldbe from about 10 mg to about 200 mg per day, in other embodiments,between about 10 mg and about 150 mg per day, in further embodiments,between about 25 and about 100 mg per day. It may be necessary to usedosages of the active ingredient outside the ranges disclosed herein insome cases, as will be apparent to those of ordinary skill in the art.Furthermore, it is noted that the clinician or treating physician willknow how and when to interrupt, adjust, or terminate therapy inconjunction with subject response.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the herein described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In certain embodiment, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate a disorder, or one or more symptoms thereof in asubject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. Inanother embodiment, the dosage of the composition or a compositionprovided herein administered to prevent, treat, manage, or ameliorate adisorder, or one or more symptoms thereof in a subject is a unit dose of0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg,1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In certain embodiments, treatment or prevention can be initiated withone or more loading doses of a compound or composition provided hereinfollowed by one or more maintenance doses. In such embodiments, theloading dose can be, for instance, about 60 to about 400 mg per day, orabout 100 to about 200 mg per day for one day to five weeks. The loadingdose can be followed by one or more maintenance doses. In certainembodiments, each maintenance does is, independently, about from about10 mg to about 200 mg per day, between about 25 mg and about 150 mg perday, or between about 25 and about 80 mg per day. Maintenance doses canbe administered daily and can be administered as single doses, or asdivided doses.

In certain embodiments, a dose of a compound or composition providedherein can be administered to achieve a steady-state concentration ofthe active ingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. Incertain embodiments, a sufficient amount of a compound or compositionprovided herein is administered to achieve a steady-state concentrationin blood or serum of the subject of from about 300 to about 4000 ng/mL,from about 400 to about 1600 ng/mL, or from about 600 to about 1200ng/mL. In some embodiments, loading doses can be administered to achievesteady-state blood or serum concentrations of about 1200 to about 8000ng/mL, or about 2000 to about 4000 ng/mL for one to five days. Incertain embodiments, maintenance doses can be administered to achieve asteady-state concentration in blood or serum of the subject of fromabout 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, orfrom about 600 to about 1200 ng/mL.

In certain embodiments, administration of the same composition may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months. In other embodiments, administration of thesame prophylactic or therapeutic agent may be repeated and theadministration may be separated by at least at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months.

In certain aspects, provided herein are unit dosages comprising acompound, or a pharmaceutically acceptable salt thereof, in a formsuitable for administration. Such forms are described in detail herein.In certain embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250mg or 10 to 50 mg active ingredient. In particular embodiments, the unitdosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mgactive ingredient. Such unit dosages can be prepared according totechniques familiar to those of skill in the art.

The dosages of the second agents are to be used in the combinationtherapies provided herein. In certain embodiments, dosages lower thanthose which have been or are currently being used to prevent or treatHCV infection are used in the combination therapies provided herein. Therecommended dosages of second agents can be obtained from the knowledgeof those of skill. For those second agents that are approved forclinical use, recommended dosages are described in, for example, Hardmanet al., eds., 1996, Goodman & Gilman's The Pharmacological Basis OfBasis Of Therapeutics 9^(th) Ed, Mc-Graw-Hill, New York; Physician'sDesk Reference (PDR) 57^(th) Ed., 2003, Medical Economics Co., Inc.,Montvale, N.J., which are incorporated herein by reference in itsentirety.

In various embodiments, the therapies (e.g., a compound provided hereinand the second agent) are administered less than 5 minutes apart, lessthan 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hoursapart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hoursto 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hoursapart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96hours to 120 hours apart. In various embodiments, the therapies areadministered no more than 24 hours apart or no more than 48 hours apart.In certain embodiments, two or more therapies are administered withinthe same patient visit. In other embodiments, the compound providedherein and the second agent are administered concurrently.

In other embodiments, the compound provided herein and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain embodiments, administration of the same agent may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In other embodiments, administration of the sameagent may be repeated and the administration may be separated by atleast at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days,45 days, 2 months, 75 days, 3 months, or 6 months.

In certain embodiments, a compound provided herein and a second agentare administered to a patient, for example, a mammal, such as a human,in a sequence and within a time interval such that the compound providedherein can act together with the other agent to provide an increasedbenefit than if they were administered otherwise. For example, thesecond active agent can be administered at the same time or sequentiallyin any order at different points in time; however, if not administeredat the same time, they should be administered sufficiently close in timeso as to provide the desired therapeutic or prophylactic effect. Incertain embodiments, the compound provided herein and the second activeagent exert their effect at times which overlap. Each second activeagent can be administered separately, in any appropriate form and by anysuitable route. In other embodiments, the compound provided herein isadministered before, concurrently or after administration of the secondactive agent.

In certain embodiments, the compound provided herein and the secondagent are cyclically administered to a patient. Cycling therapy involvesthe administration of a first agent (e.g., a first prophylactic ortherapeutic agents) for a period of time, followed by the administrationof a second agent and/or third agent (e.g., a second and/or thirdprophylactic or therapeutic agents) for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In certain embodiments, the compound provided herein and the secondactive agent are administered in a cycle of less than about 3 weeks,about once every two weeks, about once every 10 days or about once everyweek. One cycle can comprise the administration of a compound providedherein and the second agent by infusion over about 90 minutes everycycle, about 1 hour every cycle, about 45 minutes every cycle. Eachcycle can comprise at least 1 week of rest, at least 2 weeks of rest, atleast 3 weeks of rest. The number of cycles administered is from about 1to about 12 cycles, more typically from about 2 to about 10 cycles, andmore typically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecompound provided herein can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with the compoundprovided herein. In certain embodiments, the compound provided herein isadministered concurrently with one or more second agents in the samepharmaceutical composition. In another embodiment, a compound providedherein is administered concurrently with one or more second agents inseparate pharmaceutical compositions. In still another embodiment, acompound provided herein is administered prior to or subsequent toadministration of a second agent. Also contemplated are administrationof a compound provided herein and a second agent by the same ordifferent routes of administration, e.g., oral and parenteral. Incertain embodiments, when the compound provided herein is administeredconcurrently with a second agent that potentially produces adverse sideeffects including, but not limited to, toxicity, the second active agentcan advantageously be administered at a dose that falls below thethreshold that the adverse side effect is elicited.

Kits

Also provided are kits for use in methods of treatment of a liverdisorder such as HCV infections. The kits can include a compound orcomposition provided herein, a second agent or composition, andinstructions providing information to a health care provider regardingusage for treating the disorder. Instructions may be provided in printedform or in the form of an electronic medium such as a floppy disc, CD,or DVD, or in the form of a website address where such instructions maybe obtained. A unit dose of a compound or composition provided herein,or a second agent or composition, can include a dosage such that whenadministered to a subject, a therapeutically or prophylacticallyeffective plasma level of the compound or composition can be maintainedin the subject for at least 1 days. In some embodiments, a compound orcomposition can be included as a sterile aqueous pharmaceuticalcomposition or dry powder (e.g., lyophilized) composition.

In some embodiments, suitable packaging is provided. As used herein,“packaging” includes a solid matrix or material customarily used in asystem and capable of holding within fixed limits a compound providedherein and/or a second agent suitable for administration to a subject.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. If e-beam sterilizationtechniques are employed, the packaging should have sufficiently lowdensity to permit sterilization of the contents.

Methods of Use

In certain embodiments, provided herein are methods for the treatmentand/or prophylaxis of a host infected with Flaviviridae that includesthe administration of an effective amount of a compounds providedherein, or a pharmaceutically acceptable salt thereof. In certainembodiments, provided herein are methods for treating an HCV infectionin a subject. In certain embodiments, the methods encompass the step ofadministering to the subject in need thereof an amount of a compoundeffective for the treatment or prevention of an HCV infection incombination with a second agent effective for the treatment orprevention of the infection. The compound can be any compound asdescribed herein, and the second agent can be any second agent describedin the art or herein. In certain embodiments, the compound is in theform of a pharmaceutical composition or dosage form, as describedelsewhere herein.

Flaviviridae that can be treated are discussed generally in FieldsVirology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 31, 1996. In aparticular embodiment of the invention, the Flaviviridae is HCV. In analternate embodiment of the invention, the Flaviviridae is a flavivirusor pestivirus. Specific flaviviruses include, without limitation:Absettarov, Alfuy, Apoi, Aroa, Bagaza, Banzi, Bouboui, Bussuquara,Cacipacore, Carey Island, Dakar bat, Dengue 1, Dengue 2, Dengue 3,Dengue 4, Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr,Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis, Jugra,Jutiapa, Kadam, Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin,Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc, Montanamyotis leukoencephalitis, Murray valley encephalitis, Naranjal, Negishi,Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Rio Bravo,Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya, St. Louisencephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk,Spondweni, Stratford, Tembusu, Tyuleniy, Uganda S, Usutu, Wesselsbron,West Nile, Yaounde, Yellow fever, and Zika.

Pestiviruses that can be treated are discussed generally in FieldsVirology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 33, 1996.Specific pestiviruses include, without limitation: bovine viral diarrheavirus (“BVDV”), classical swine fever virus (“CSFV,” also called hogcholera virus), and border disease virus (“BDV”).

In certain embodiments, the subject can be any subject infected with, orat risk for infection with, HCV. Infection or risk for infection can bedetermined according to any technique deemed suitable by thepractitioner of skill in the art. In certain embodiments, subjects arehumans infected with HCV.

In certain embodiments, the subject has never received therapy orprophylaxis for an HCV infection. In further embodiments, the subjecthas previously received therapy or prophylaxis for an HCV infection. Forinstance, in certain embodiments, the subject has not responded to anHCV therapy. For example, under current interferon therapy, up to 50% ormore HCV subjects do not respond to therapy. In certain embodiments, thesubject can be a subject that received therapy but continued to sufferfrom viral infection or one or more symptoms thereof. In certainembodiments, the subject can be a subject that received therapy butfailed to achieve a sustained virologic response. In certainembodiments, the subject has received therapy for an HCV infection buthas failed to show, for example, a 2 log₁₀ decline in HCV RNA levelsafter 12 weeks of therapy. It is believed that subjects who have notshown more than 2 log₁₀ reduction in serum HCV RNA after 12 weeks oftherapy have a 97-100% chance of not responding.

In certain embodiments, the subject is a subject that discontinued anHCV therapy because of one or more adverse events associated with thetherapy. In certain embodiments, the subject is a subject where currenttherapy is not indicated. For instance, certain therapies for HCV areassociated with neuropsychiatric events. Interferon (IFN)-alfa plusribavirin is associated with a high rate of depression. Depressivesymptoms have been linked to a worse outcome in a number of medicaldisorders. Life-threatening or fatal neuropsychiatric events, includingsuicide, suicidal and homicidal ideation, depression, relapse of drugaddiction/overdose, and aggressive behavior have occurred in subjectswith and without a previous psychiatric disorder during HCV therapy.Interferon-induced depression is a limitation for the treatment ofchronic hepatitis C, especially for subjects with psychiatric disorders.Psychiatric side effects are common with interferon therapy andresponsible for about 10% to 20% of discontinuations of current therapyfor HCV infection.

Accordingly, provided are methods of treating or preventing an HCVinfection in subjects where the risk of neuropsychiatric events, such asdepression, contraindicates treatment with current HCV therapy. Incertain embodiments, provided are methods of treating or preventing HCVinfection in subjects where a neuropsychiatric event, such asdepression, or risk of such indicates discontinuation of treatment withcurrent HCV therapy. Further provided are methods of treating orpreventing HCV infection in subjects where a neuropsychiatric event,such as depression, or risk of such indicates dose reduction of currentHCV therapy.

Current therapy is also contraindicated in subjects that arehypersensitive to interferon or ribavirin, or both, or any othercomponent of a pharmaceutical product for administration of interferonor ribavirin. Current therapy is not indicated in subjects withhemoglobinopathies (e.g., thalassemia major, sickle-cell anemia) andother subjects at risk from the hematologic side effects of currenttherapy. Common hematologic side effects include bone marrowsuppression, neutropenia and thrombocytopenia. Furthermore, ribavirin istoxic to red blood cells and is associated with hemolysis. Accordingly,in certain embodiments, provided are methods of treating or preventingHCV infection in subjects hypersensitive to interferon or ribavirin, orboth, subjects with a hemoglobinopathy, for instance thalassemia majorsubjects and sickle-cell anemia subjects, and other subjects at riskfrom the hematologic side effects of current therapy.

In certain embodiments, the subject has received an HCV therapy anddiscontinued that therapy prior to administration of a method providedherein. In further embodiments, the subject has received therapy andcontinues to receive that therapy along with administration of a methodprovided herein. The methods can be co-administered with other therapyfor HBC and/or HCV according to the judgment of one of skill in the art.In certain embodiments, the methods or compositions provided herein canbe co-administered with a reduced dose of the other therapy for HBCand/or HCV.

In certain embodiments, provided are methods of treating a subject thatis refractory to treatment with interferon. For instance, in someembodiments, the subject can be a subject that has failed to respond totreatment with one or more agents selected from the group consisting ofinterferon, interferon α, pegylated interferon α, interferon plusribavirin, interferon α plus ribavirin and pegylated interferon α plusribavirin. In some embodiments, the subject can be a subject that hasresponded poorly to treatment with one or more agents selected from thegroup consisting of interferon, interferon α, pegylated interferon α,interferon plus ribavirin, interferon α plus ribavirin and pegylatedinterferon α plus ribavirin. A pro-drug form of ribavirin, such astaribavirin, may also be used.

In certain embodiments, the subject has, or is at risk for, co-infectionof HCV with HIV. For instance, in the United States, 30% of HIV subjectsare co-infected with HCV and evidence indicates that people infectedwith HIV have a much more rapid course of their hepatitis C infection.Maier and Wu, 2002, World J Gastroenterol 8:577-57. The methods providedherein can be used to treat or prevent HCV infection in such subjects.It is believed that elimination of HCV in these subjects will lowermortality due to end-stage liver disease. Indeed, the risk ofprogressive liver disease is higher in subjects with severeAIDS-defining immunodeficiency than in those without. See, e.g., Lesenset al., 1999, J Infect Dis 179:1254-1258. In certain embodiments,compounds provided herein have been shown to suppress HIV in HIVsubjects. Thus, in certain embodiments, provided are methods of treatingor preventing HIV infection and HCV infection in subjects in needthereof.

In certain embodiments, the compounds or compositions are administeredto a subject following liver transplant. Hepatitis C is a leading causeof liver transplantation in the U.S., and many subjects that undergoliver transplantation remain HCV positive following transplantation. Incertain embodiments, provided are methods of treating such recurrent HCVsubjects with a compound or composition provided herein. In certainembodiments, provided are methods of treating a subject before, duringor following liver transplant to prevent recurrent HCV infection.

Assay Methods

Compounds can be assayed for HCV activity according to any assay knownto those of skill in the art.

Further, compounds can be assayed for accumulation in liver cells of asubject according to any assay known to those of skill in the art. Incertain embodiments, a compound can be administered to the subject, anda liver cell of the subject can be assayed for the compound or aderivative thereof, e.g. a nucleoside, nucleoside phosphate ornucleoside triphosphate derivative thereof.

In certain embodiments, a 2′-chloro nucleoside analog compound isadministered to cells, such as liver cells, in vivo or in vitro, and thenucleoside triphosphate levels delivered intracellularly are measured,to indicate delivery of the compound and triphosphorylation in the cell.The levels of intracellular nucleoside triphosphate can be measuredusing analytical techniques known in the art. Methods of detecting ddATPare described herein below by way of example, but other nucleosidetriphosphates can be readily detected using the appropriate controls,calibration samples and assay techniques.

In certain embodiments, ddATP concentrations are measured in a sample bycomparison to calibration standards made from control samples. The ddATPconcentrations in a sample can be measured using an analytical methodsuch as HPLC LC MS. In certain embodiments, a test sample is compared toa calibration curve created with known concentrations of ddATP tothereby obtain the concentration of that sample.

In certain embodiments, the samples are manipulated to remove impuritiessuch as salts (Na⁺, K⁺, etc.) before analysis. In certain embodiments,the lower limit of quantitation is about ˜0.2 pmol/mL for hepatocytecellular extracts particularly where reduced salt is present.

In certain embodiments, the method allows successfully measuringtriphosphate nucleotides formed at levels of 1-10,000 pmol per millioncells in e.g. cultured hepatocytes and HepG2 cells.

Second Therapeutic Agents

In certain embodiments, the compounds and compositions provided hereinare useful in methods of treatment of a liver disorder, that comprisefurther administration of a second agent effective for the treatment ofthe disorder, such as HCV infection in a subject in need thereof. Thesecond agent can be any agent known to those of skill in the art to beeffective for the treatment of the disorder, including those currentlyapproved by the FDA.

In certain embodiments, a compound provided herein is administered incombination with one second agent. In further embodiments, a secondagent is administered in combination with two second agents. In stillfurther embodiments, a second agent is administered in combination withtwo or more second agents.

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound provided herein)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” includes a combination of acompound provided herein and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent) and/or to administer said therapyless frequently reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

The active compounds provided herein can be administered in combinationor alternation with another therapeutic agent, in particular an anti-HCVagent. In combination therapy, effective dosages of two or more agentsare administered together, whereas in alternation or sequential-steptherapy, an effective dosage of each agent is administered serially orsequentially. The dosages given will depend on absorption, inactivationand excretion rates of the drug as well as other factors known to thoseof skill in the art. It is to be noted that dosage values will also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimens andschedules should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions. In certain embodiments, ananti-HCV (or anti-pestivirus or anti-flavivirus) compound that exhibitsan EC₅₀ of 10-15 μM. In certain embodiments, less than 1-5 μM, isdesirable.

It has been recognized that drug-resistant variants of flaviviruses,pestiviruses or HCV can emerge after prolonged treatment with anantiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in viral replication. The efficacyof a drug against the viral infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

Any of the viral treatments described in the Background of the Inventioncan be used in combination or alternation with the compounds describedin this specification. Non-limiting examples of second agents include:

HCV Protease inhibitors: Examples include Medivir HCV Protease Inhibitor(HCV-PI or TMC435) (Medivir/Tibotec); MK-7009 (Merck), RG7227 (ITMN-191)(Roche/Pharmasset/InterMune), boceprevir (SCH 503034) (Schering), SCH446211 (Schering), narlaprevir SCH900518 (Schering/Merck), ABT-450(Abbott/Enanta), ACH-1625 (Achillion), BI 201335 (Boehringer Ingelheim),PHX1766 (Phenomix), VX-500 (Vertex) and telaprevir (VX-950) (Vertex).Further examples of protease inhibitors include substrate-based NS3protease inhibitors (Attwood et al., Antiviral peptide derivatives, PCTWO 98/22496, 1998; Attwood et al., Antiviral Chemistry and Chemotherapy1999, 10, 259-273; Attwood et al., Preparation and use of amino acidderivatives as anti-viral agents, German Patent Pub. DE 19914474; Tunget al., Inhibitors of serine proteases, particularly hepatitis C virusNS3 protease, PCT WO 98/17679), including alphaketoamides andhydrazinoureas, and inhibitors that terminate in an electrophile such asa boronic acid or phosphonate (Llinas-Brunet et al, Hepatitis Cinhibitor peptide analogues, PCT WO 99/07734); Non-substrate-based NS3protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamidederivatives (Sudo K. et al., Biochemical and Biophysical ResearchCommunications, 1997, 238, 643-647; Sudo K. et al., Antiviral Chemistryand Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, theformer substituted on the amide with a 14 carbon chain and the latterprocessing a para-phenoxyphenyl group; and Sch 68631, aphenanthrenequinone, an HCV protease inhibitor (Chu M. et al.,Tetrahedron Letters 37:7229-7232, 1996).

SCH 351633, isolated from the fungus Penicillium griseofulvum, wasidentified as a protease inhibitor (Chu M. et al., Bioorganic andMedicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36:1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCVinclude, for example, U.S. Pat. No. 6,004,933 to Spruce et al., whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al., whichdiscloses synthetic inhibitors of hepatitis C virus NS3 protease; U.S.Pat. No. 5,538,865 to Reyes et a; WO 02/008251 to Corvas International,Inc., and U.S. Pat. No. 7,169,760, US2005/176648, WO 02/08187 and WO02/008256 to Schering Corporation. HCV inhibitor tripeptides aredisclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 toBoehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb. Diarylpeptides as NS3 serine protease inhibitors of HCV are disclosed in WO02/48172 and U.S. Pat. No. 6,911,428 to Schering Corporation.Imidazoleidinones as NS3 serine protease inhibitors of HCV are disclosedin WO 02/08198 and U.S. Pat. No. 6,838,475 to Schering Corporation andWO 02/48157 and U.S. Pat. No. 6,727,366 to Bristol Myers Squibb. WO98/17679 and U.S. Pat. No. 6,265,380 to Vertex Pharmaceuticals and WO02/48116 and U.S. Pat. No. 6,653,295 to Bristol Myers Squibb alsodisclose HCV protease inhibitors. Further examples of HCV serineprotease inhibitors are provided in U.S. Pat. No. 6,872,805(Bristol-Myers Squibb); WO 2006000085 (Boehringer Ingelheim); U.S. Pat.No. 7,208,600 (Vertex); US 2006/0046956 (Schering-Plough); WO2007/001406 (Chiron); US 2005/0153877; WO 2006/119061 (Merck); WO00/09543 (Boehringer Ingelheim), U.S. Pat. No. 6,323,180 (BoehringerIngelheim) WO 03/064456 (Boehringer Ingelheim), U.S. Pat. No. 6,642,204(Boehringer Ingelheim), WO 03/064416 (Boehringer Ingelheim), U.S. Pat.No. 7,091,184 (Boehringer Ingelheim), WO 03/053349 (Bristol-MyersSquibb), U.S. Pat. No. 6,867,185, WO 03/099316 (Bristol-Myers Squibb),U.S. Pat. No. 6,869,964, WO 03/099274 (Bristol-Myers Squibb), U.S. Pat.No. 6,995,174, WO 2004/032827 (Bristol-Myers Squibb), U.S. Pat. No.7,041,698, WO 2004/043339 and U.S. Pat. No. 6,878,722 (Bristol-MyersSquibb).

Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18),especially compound RD-1-6250, possessing a fused cinnamoyl moietysubstituted with a long alkyl chain, RD4 6205 and RD4 6193;

Thiazolidines and benzanilides identified in Kakiuchi N. et al., J. EBSLetters 421, 217-220; Takeshita N. et al., Analytical Biochemistry,1997, 247, 242-246;

A phenanthrenequinone possessing activity against protease in a SDS-PAGEand autoradiography assay isolated from the fermentation culture brothof Streptomyces sp., SCH 68631 (Chu M. et al., Tetrahedron Letters,1996, 37, 7229-7232), and SCH 351633, isolated from the fungusPenicillium griseofulvum, which demonstrates activity in a scintillationproximity assay (Chu M. et al., Bioorganic and Medicinal ChemistryLetters 9, 1949-1952);

Helicase inhibitors (Diana G. D. et al., Compounds, compositions andmethods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al., Piperidine derivatives, pharmaceutical compositions thereofand their use in the treatment of hepatitis C, PCT WO 97/36554);

HCV polymerase inhibitors, including nucleoside and non-nucleosidepolymerase inhibitors, such as ribavirin, viramidine, clemizole,filibuvir (PF-00868554), HCV POL, NM 283 (valopicitabine), MK-0608,7-Fluoro-MK-0608, MK-3281, IDX-375, ABT-072, ABT-333, ANA598, BI 207127,GS 9190, PSI-6130, R1626, PSI-6206, PSI-938, PSI-7851, PSI-7977, RG1479,RG7128, HCV-796 VCH-759 or VCH-916.

Gliotoxin (Ferrari R. et al., Journal of Virology, 1999, 73, 1649-1654),and the natural product cerulenin (Lohmann V. et al., Virology, 1998,249, 108-118);

Interfering RNA (iRNA) based antivirals, including short interfering RNA(siRNA) based antivirals, such as Sirna-034 and others described inInternational Patent Publication Nos. WO/03/070750 and WO 2005/012525,and US Patent Publication No. US 2004/0209831.

Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementaryto sequence stretches in the 5′ non-coding region (NCR) of the virus(Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides 326-348comprising the 3′ end of the NCR and nucleotides 371-388 located in thecore coding region of the HCV RNA (Alt M. et al., Archives of Virology,1997, 142, 589-599; Galderisi U. et al., Journal of Cellular Physiology,1999, 181, 251-257);

Inhibitors of IRES-dependent translation (Ikeda N et al., Agent for theprevention and treatment of hepatitis C, Japanese Patent Pub.JP-08268890; Kai Y. et al., Prevention and treatment of viral diseases,Japanese Patent Pub. JP-10101591);

HCV NS5A inhibitors, such as BMS-790052 (daclatasvir, Bristol-MyersSquibb), PPI-461 (Presidio Pharmaceuticals), PPI-1301 (PresidioPharmaceuticals), IDX-719 (Idenix Pharmaceuticals), AZD7295 (ArrowTherapeutics, AstraZeneca), EDP-239 (Enanta), ACH-2928 (Achillion),ACH-3102 (Achillion), ABT-267 (Abbott), or GS-5885 (Gilead);

HCV entry inhibitors, such as celgosivir (MK-3253) (MIGENIX Inc.), SP-30(Samaritan Pharmaceuticals), ITX4520 (iTherX), ITX5061 (iTherX), PRO-206(Progenics Pharmaceuticals) and other entry inhibitors by ProgenicsPharmaceuticals, e.g., as disclosed in U.S. Patent Publication No.2006/0198855.

Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D. J. et al.,Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat. No.6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and 5,610,054to Draper et al.; and

Nucleoside analogs have also been developed for the treatment ofFlaviviridae infections.

In certain embodiments, the compounds provided herein can beadministered in combination with any of the compounds described byIdenix Pharmaceuticals in International Publication Nos. WO 01/90121, WO01/92282, WO 2004/003000, 2004/002422 and WO 2004/002999.

Other patent applications disclosing the use of certain nucleosideanalogs that can be used as second agents to treat hepatitis C virusinclude: PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) andPCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by BioChemPharma, Inc. (now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425;filed Jan. 18, 2002); PCT/US02/03086 (WO 02/057287; filed Jan. 18,2002); U.S. Pat. Nos. 7,202,224; 7,125,855; 7,105,499 and 6,777,395 byMerck & Co., Inc.; PCT/EP01/09633 (WO 02/18404; published Aug. 21,2001); US 2006/0040890; 2005/0038240; 2004/0121980; U.S. Pat. Nos.6,846,810; 6,784,166 and 6,660,721 by Roche; PCT Publication Nos. WO01/79246 (filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18, 2001) and WO02/48165; US 2005/0009737; US 2005/0009737; U.S. Pat. Nos. 7,094,770 and6,927,291 by Pharmasset, Ltd.

Further compounds that can be used as second agents to treat hepatitis Cvirus are disclosed in PCT Publication No. WO 99/43691 to EmoryUniversity, entitled “2′-Fluoronucleosides”. The use of certain2′-fluoronucleosides to treat HCV is disclosed.

Other compounds that can be used as second agents include1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E andother antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.),squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki etal.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 toDiana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Dianaet al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wanget al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan etal.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidines(U.S. Pat. No. 5,830,905 to Diana et al.).

In certain embodiments, a compound of a formula provided herein, or acomposition comprising a compound of a formula provided herein, isadministered in combination or alternation with a second anti-viralagent selected from the group consisting of an interferon, a nucleotideanalogue, a polymerase inhibitor, an NS3 protease inhibitor, an NS5Ainhibitor, an entry inhibitor, a non-nucleoside polymerase inhibitor, acyclosporine immune inhibitor, an NS4A antagonist, an NS4B-RNA bindinginhibitor, a locked nucleic acid mRNA inhibitor, a cyclophilininhibitor, and combinations thereof.

Exemplary Second Therapeutic Agents for Treatment of HCV

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus interferon, such as Intron A® (interferon alfa-2b) and; Roferon A®(Recombinant interferon alfa-2a), Infergen® (consensus interferon;interferon alfacon-1), PEG-Intron® (pegylated interferon alfa-2b), andPegasys® (pegylated interferon alfa-2a). In certain embodiments, one ormore compounds provided herein can be administered in combination oralternation with ribavirin and in combination or alternation with ananti-hepatitis C virus interferon. In certain embodiments, one or morecompounds provided herein can be administered in combination oralternation with ribavirin, in combination or alternation with ananti-hepatitis C virus interferon, and in combination or alternationwith an anti-hepatitis C virus protease inhibitor. In certainembodiments, one or more compounds provided herein can be administeredin combination or alternation with ribavirin. In certain embodiments,one or more compounds provided herein can be administered in combinationor alternation with an anti-hepatitis C virus interferon and withoutribavirin. In certain embodiments, one or more compounds provided hereincan be administered in combination or alternation with an anti-hepatitisC virus interferon, in combination or alternation with an anti-hepatitisC virus protease inhibitor, and without ribavirin.

In certain embodiments, the anti-hepatitis C virus interferon isinfergen, IL-29 (PEG-Interferon lambda), R7025 (Maxy-alpha), Belerofon,Oral Interferon alpha, BLX-883 (Locteron), omega interferon, multiferon,medusa interferon, Albuferon or REBIF®.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus polymerase inhibitor, such as ribavirin, viramidine, HCV POL, NM283 (valopicitabine), MK-0608, 7-Fluoro-MK-0608, PSI-6130, R1626,PSI-6206, PSI-938, R1479, HCV-796, VX-950 (Telaprevir, Vertex), GS 9190NN (Gilead), GS 9256 (Gilead), PSI-7792 (BMS), BI 207127 (BI), R7128(Roche), or PSI-7977 (Pharmasset), PSI-938 (Pharmasset), VX-222(Vertex), ALS-2200 (Vertex), ALS-2158 (Vertex), MK-0608 (Merck),TMC649128 (Medivir), PF-868554 (Pfizer), PF-4878691 (Pfizer), ANA598(Roche), VCH-759 (Vertex), IDX184 (Idenix), IDX375 (Idenix), A-837093(Abbott), GS 9190 (Gilead), GSK625433 (GlaxoSmithKline), ABT-072(Abbott), ABT-333 (Abbott), INX-189 (Inhibitex), or EDP-239 (Enanta).

In certain embodiments, the one or more compounds provided herein can beadministered in combination with ribavarin and an anti-hepatitis C virusinterferon, such as Intron A® (interferon alfa-2b) and Pegasys®(Peginterferon alfa-2a); Roferon A® (Recombinant interferon alfa-2a),Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron®(pegylated interferon alfa-2b), Zalbin (albinterferon alfa-2b), omegainterferon, pegylated interferon lambda, and Pegasys® (pegylatedinterferon alfa-2a).

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus protease inhibitor such as ITMN-191, SCH 503034 (bocepravir),VX950 (telaprevir), VX985, VX500, VX813, PHX1766, BMS-650032, GS 9256,BI 201335, IDX320, R7227, MK-7009 (vaniprevir), TMC435, BMS-791325,ACH-1625, ACH-2684, ABT-450, or AVL-181.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an HCV NS5A inhibitor,such as BMS-790052 (daclatasvir, Bristol-Myers Squibb), PPI-461(Presidio Pharmaceuticals), PPI-1301 (Presidio Pharmaceuticals), IDX-719(Idenix Pharmaceuticals), AZD7295 (Arrow Therapeutics, AstraZeneca),EDP-239 (Enanta), ACH-2928 (Achillion), ACH-3102 (Achillion), ABT-267(Abbott), or GS-5885 (Gilead).

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus vaccine, such as TG4040, PeviPROTM, CGI-5005, HCV/MF59, GV1001,IC41, GNI-103, GenPhar HCV vaccine, C-Vaxin, CSL123, Hepavaxx C,ChronVac-C® or INNO0101 (E1).

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus monoclonal antibody, such as MBL-HCV1, AB68 or XTL-6865 (formerlyHepX-C); or an anti-hepatitis C virus polyclonal antibody, such ascicavir.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus immunomodulator, such as Zadaxin® (thymalfasin), SCV-07, NOV-205or Oglufanide.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with cyclophilin inhibitor,such as Enanta cyclophilin binder, SCY-635, or Debio-025.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with Nexavar, doxorubicin,PI-88, amantadine, JBK-122, VGX-410C, MX-3253 (Ceglosivir), Suvus(BIVN-401 or virostat), PF-03491390 (formerly IDN-6556), G126270,UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ, ANA975, AVI-4065,Bavituxinab (Tarvacin), Alinia (nitrazoxanide) or PYN17.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with telaprevir, bocepravir,interferon alfacon-1, interferon alfa-2b, pegylated interferon alpha 2a,pegylated interferon alpha 2b, ribavirin, or combinations thereof.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with a protease inhibitor. Incertain embodiments, one or more compounds provided herein can beadministered in combination or alternation with telaprevir. In certainembodiments, one or more compounds provided herein can be administeredin combination or alternation with bocepravir.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with a protease inhibitor andin combination or alternation with ribavirin. In certain embodiments,one or more compounds provided herein can be administered in combinationor alternation with telaprevir and in combination or alternation withribavirin. In certain embodiments, one or more compounds provided hereincan be administered in combination or alternation with bocepravir and incombination or alternation with ribavirin.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with a protease inhibitor andnot in combination or alternation with ribavirin. In certainembodiments, one or more compounds provided herein can be administeredin combination or alternation with telaprevir and not in combination oralternation with ribavirin. In certain embodiments, one or morecompounds provided herein can be administered in combination oralternation with bocepravir and not in combination or alternation withribavirin.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an interferon. Incertain embodiments, one or more compounds provided herein can beadministered in combination or alternation with interferon alfacon-1. Incertain embodiments, one or more compounds provided herein can beadministered in combination or alternation with interferon alfa-2b. Incertain embodiments, one or more compounds provided herein can beadministered in combination or alternation with pegylated interferonalpha 2a. In certain embodiments, one or more compounds provided hereincan be administered in combination or alternation with pegylatedinterferon alpha 2b.

In certain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an interferon and incombination or alternation with ribavirin. In certain embodiments, oneor more compounds provided herein can be administered in combination oralternation with interferon alfacon-1and in combination or alternationwith ribavirin. In certain embodiments, one or more compounds providedherein can be administered in combination or alternation with interferonalfa-2b and in combination or alternation with ribavirin. In certainembodiments, one or more compounds provided herein can be administeredin combination or alternation with pegylated interferon alpha 2a and incombination or alternation with ribavirin. In certain embodiments, oneor more compounds provided herein can be administered in combination oralternation with pegylated interferon alpha 2b and in combination oralternation with ribavirin.

In certain embodiments, one or more compounds can be administered incombination or alternation with one or more of the second agentsprovided herein and not in combination or alternation with ribavirin. Incertain embodiments, one or more compounds provided herein can beadministered in combination or alternation with an interferon and not incombination or alternation with ribavirin. In certain embodiments, oneor more compounds provided herein can be administered in combination oralternation with interferon alfacon-1and not in combination oralternation with ribavirin. In certain embodiments, one or morecompounds provided herein can be administered in combination oralternation with interferon alfa-2b and not in combination oralternation with ribavirin. In certain embodiments, one or morecompounds provided herein can be administered in combination oralternation with pegylated interferon alpha 2a and not in combination oralternation with ribavirin. In certain embodiments, one or morecompounds provided herein can be administered in combination oralternation with pegylated interferon alpha 2b and not in combination oralternation with ribavirin.

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of the American ChemicalSociety or the Journal of Biological Chemistry. Specifically, butwithout limitation, the following abbreviations may be used in theexamples and throughout the specification: g (grams); mg (milligrams);mL (milliliters); μL (microliters); mM (millimolar); μM (micromolar); Hz(Hertz); MHz (megahertz); mmol (millimoles); hr or hrs (hours); min(minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC(thin layer chromatography); HPLC (high pressure liquid chromatography);THF (tetrahydrofuran); CDCl₃ (deuterated chloroform); AcOH (aceticacid); DCM (dichloromethane); DMSO (dimethylsulfoxide); DMSO-d₆(deuterated dimethylsulfoxide); EtOAc (ethyl acetate); MeOH (methanol);and BOC (t-butyloxycarbonyl).

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade). All reactions are conducted at room temperature unlessotherwise noted. Synthetic methodologies illustrated herein are intendedto exemplify the applicable chemistry through the use of specificexamples and are not indicative of the scope of the disclosure.

Example 1 Preparation of 2′-Chloro Nucleoside Analogs

Ethyl(3R)-2-chloro-3-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-hydroxy-2-methylpropanoate(A2)

A 5 L flange flask was fitted with a thermometer, nitrogen inlet,pressure equalizing dropping funnel, bubbler, and a suba•seal. Methyllithium solution (1.06 L, 1.6 M in diethylether, 1.7 equiv.) was added,and the solution was cooled to about −25° C. Diisopropyl amine (238 ml,1.7 equiv.) was added using the dropping funnel over about 40 minutes.The reaction was left stirring, allowing to warm to ambient temperatureovernight. CO_(2(s))/acetone cooling was applied to the LDA solution,cooling to about −70° C.

R-Glyceraldehyde dimethylacetal solution (50% in DCM) was evaporateddown to ˜100 mbar at a bath temp of 35° C., to remove the DCM, thenazeotroped with anhydrous hexane (200 ml), under the same Büchiconditions. ¹H NMR was used to confirm that all but a trace of DCMremained.

The fresh aldehyde (130 g, 1 mol) and ethyl 2-chloropropionionate (191ml, 1.5 equiv.) were placed in a 1 L round bottom flask, which wasfilled with toluene (800 ml). This solution was cooled in aCO_(2(s))/acetone bath, and added via cannula to the LDA solution overabout 50 minutes, keeping the internal temperature of the reactionmixture cooler than −60° C. The mixture was stirred with cooling(internal temp. slowly fell to ˜−72° C.) for 90 min, then warmed to roomtemperature over 30 minutes using a water bath. This solution was addedto a sodium dihydrogen phosphate solution equivalent to 360 g of NaH₂PO₄in 1.5 L of ice/water, over about 10 minutes, with ice-bath cooling. Themixture was stirred for 20 minutes, then transferred to a sep. funnel,and partitioned. The aqueous layer was further extracted with EtOAc (2×1L), and the combined organic extracts were dried over sodium sulfate.The volatiles were removed in vacuo (down to 20 mbar). The resultant oilwas hydrolyzed crude.

(3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one(A4)

The crude oil A2 was taken up in acetic acid (1.5 L, 66% in water) andheated to 90° C. over one hour, then at held at that temperature for onehour. Once the mixture had cooled to room temperature, the volatileswere removed in vacuo, and azeotroped with toluene (500 ml). Theresultant oil was combined with some mixed material from an earliersynthesis and columned in two portions (each ˜1.25 L of silica, 38→75%EtOAc in DCM). The lower of the two main spots is the desired material;fractions containing this material as the major component were combinedand the solvent removed in vacuo to give 82 g of orange solid whose ¹HNMR showed the material to be of about 57% purity (of the remainder 29%was the indicated epimer). This material was recrystallized fromtoluene/butanone (600 ml/˜185 ml), the butanone being the ‘good’solvent. The resultant solid was filtered washing with toluene andhexane, and dried in vacuo to give product of about 92% purity (30 g).

(2R,3R,4R)-2-[(benzoyloxy)methyl]-4-chloro-4-methyl-5-oxooxolan-3-ylbenzoate (A5)

A 2 L 3-neck round bottom flask was fitted with an overhead stirrer,thermometer and pressure equalizing dropping funnel (→N₂). Theintermediate A4 (160 mmol) in acetonitrile (1 L) was added, followed by4-dimethylaminopyridine (3.2 mmol) and benzoyl chloride (352 mmol).Finally triethylamine (384 mmol) was added over 10 minutes using thedropping funnel. The addition of the triethylamine is accompanied by amild exotherm, which obviated the addition of a cold water bath to keepthe internal temperature below 25° C. The reaction was stirred atambient temperature for 2.5 hours. The reaction mixture was transferredto a sep. funnel with EtOAc (2 L) and half saturated brine (2 L), andpartitioned. The aqueous layer was re-extracted with EtOAc (1 L). Thecombined organic layers were washed with 50% sodium bicarbonate/25%brine (1.5 L) and dried over sodium sulfate, to give 62 g of solid. Thiswas recrystallized from 1.8 L of 1:1 toluene/trimethylpentane (95° C.),to give 52.4 g of product.

¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.91 (s, 3H), 4.57 (dd, J=5.12 Hz andJ=12.57 Hz, 1H), 4.77 (dd, J=3.29 Hz and J=12.68 Hz, 1H), 4.92-4.96 (m,1H), 5.60 (d, J=8.36 Hz, 1H), 7.38-7.66 (m, 6H), 7.97-7.99 (m, 2H),8.08-8.10 (m, 2H); MS (ESI) m/z=411.1 (MNa⁺).

3,5-Di-O-benzoyl-2-C-chloro-2-C-methyl-D-ribofuranose (A6)

To a solution of A5 (14.48 mmol) in anhydrous tetrahydrofurane (70 ml)was added under inert atmosphere at −35° C., LiAlH(OtBu)₃ (1M intetrahydrofurane, 21.7 mmol) over a 30 min period. The reaction mixturewas stirred for 1 hour at −20° C. and quenched by addition of asaturated NH₄Cl solution, keeping the temperature bellow 0° C. Ethylacetate was added and the white suspension was filtered through a pad ofcelite and washed with ethyl acetate. The filtrate was extracted withethyl acetate twice. The combined organic layers were dried overanhydrous sodium sulfate, filtered and evaporated under reducedpressure. The residue was purified by chromatography on silica gel(eluent: petroleum ether/ethyl acetate 0 to 20%). The product was driedin vacuum (50° C.) overnight to afford expected intermediate as acolorless oil in 96% yield (mixture α/β: 45/55).

¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.74 (s, 1.75H_(β)), 1.76 (s,1.25H_(α)), 4.42-4.69 (m, 3H), 5.30 (d, J=12.8 Hz, 0.55H_(β)), 5.43-5.47(m, 0.45H_(α)), 5.60 (d, J=7.0 Hz, 0.55H_(β)), 5.78 (d, J=7.0 Hz,0.45H_(α)), 7.35-7.41 (m, 2H), 7.45-7.56 (m, 3H), 7.59-7.65 (m, 1H),7.96-8.04 (m, 2H), 8.06-8.14 (m, 2H); MS (ESI) m/z=413 (MNa⁺).

3,5-Di-O-benzoyl-2-C-chloro-2-C-methyl-D-arabinofuranosyl bromide (A7)

To a solution of A6 (12.80 mmol) in anhydrous dichloromethane (80 ml)was added under inert atmosphere at −20° C., triphenylphosphine (18.0mmol). The reaction mixture was stirred for 15 minutes at −20° C. andCBr₄ (19.20 mmol) was added. The reaction mixture was then stirred for 1hour at −20° C. The crude was partially concentrated under reducedpressure (bath temperature bellow 30° C.) and directly purified bychromatography on silica gel (eluent: petroleum ether/ethyl acetate 0 to30%) to afford a mixture of β sugar A7a (1.67 g) and α sugar A7b (2.15g) as a colorless gum in 66% global yield.

¹H NMR (CDCl₃, 400 MHz): β sugar δ (ppm) 1.93 (s, 3H), 4.60-4.88 (m,3H), 6.08 (d, J=7.9 Hz, 1H), 6.62 (s, 1H), 7.31-7.38 (m, 2H), 7.41-7.55(m, 3H), 7.59-7.65 (m, 1H), 8.00-8.05 (m, 2H), 8.06-8.12 (m, 2H); αsugar δ (ppm) 1.88 (s, 3H), 4.66-4.89 (m, 3H), 5.37 (d, J=4.88 Hz, 1H),6.44 (s, 1H), 7.41-7.55 (m, 4H), 7.54-7.65 (m, 2H), 8.00-8.05 (m, 2H),8.14-8.20 (m, 2H); MS (ESI) m/z=476/478 (MNa⁺).

3′,5′-Di-O-benzoyl-2′-C-chloro-2′-C-methyl-4-benzoyl-cytidine (A8)

To a suspension of N-benzoyl cytosine (9.48 mmol), and a catalyticamount of ammonium sulfate in 4-chlorobenzene (24 ml) was added HMDS(28.44 mmol). The reaction mixture was heated during 2 hours at 140° C.The solvent was removed under inert atmosphere and the residue was takenin 4-chlorobenzene (15 ml). Then, A7b (4.74 mmol) in chlorobenzene (10ml) was added dropwise to the reaction mixture followed by SnCl₄ (14.22mmol) dropwise. The reaction mixture was stirred at 70° C. overnight,cooled to room temperature and diluted with dichloromethane and asaturated NaHCO₃ solution. The white suspension was filtered through apad of celite and washed with dichloromethane. The filtrate wasextracted with dichloromethane twice. The combined organic layers weredried over anhydrous Na₂SO₄, filtered and evaporated under reducedpressure to afford expected intermediate as a white solid in 89% yield.

¹H NMR (DMSO, 400 MHz): δ (ppm) 1.58 (s, 3H), 4.68-4.81 (m, 3H), 5.68(brs, 1H), 6.55 (brs, 1H), 7.36 (d, J=7.84 Hz, 1H), 7.39-7.76 (m, 9H),7.88-8.07 (m, 6H), 8.30 (d, J=7.84 Hz, 1H); MS (ESI) m/z=588 (MH⁺).

3′,5′-Di-O-benzoyl-2′-C-chloro-2′-C-methyluridine (A9)

A suspension of A8 (4.19 mmol) in an acetic acid/water mixture (67 ml/17ml, v/v), was heated at 110° C. for 3 hours. The reaction mixture wasevaporated to dryness and co-evaporated with toluene (three times) toafford expected intermediate in quantitative yield as an oil which wasdirectly used for the next step; MS (ESI) m/z=485 (MH⁺).

2′-C-Chloro-2′-C-methyluridine (301)

Intermediate A9 (4.19 mmol) in 7 N methanolic ammonia (80 ml) wasstirred at room temperature for 24 hours. The mixture was evaporated todryness, diluted with water and transferred into a separatory funnel.The aqueous layer was extracted with dichloromethane and water wasremoved under reduced pressure. The residue was purified by flash RP18gel chromatography (eluent: water/acetonitrile 0 to 40%) to afford pureexpected compound as a white foam in 79% yield.

¹H NMR (DMSO, 400 MHz): δ (ppm) 1.44 (s, 3H), 3.60-3.68 (m, 1H),3.80-3.94 (m, 3H), 5.39 (t, J=4.45 Hz, 1H), 5.63 (d, J=8.26 Hz, 1H),5.93 (d, J=5.72 Hz, 1H), 6.21 (s, 1H), 8.16 (d, J=8.90 Hz, 1H), 11.44(m, 1H); MS (ESI) m/z=277 (MH⁺).

General Method D

The following procedure was used to obtain intermediates A22a, A22b,A22c and A22d.

To a stirred solution of 4-nitrophenyl dichlorophosphate (Aldrich)(14.91 mmol) in DCM (30 mL) was added a solution of phenol (Aldrich)(14.91 mmol) and TEA (16.40 mmol) in DCM (30 mL) at −78° C. over aperiod of 20 minutes. The reaction mixture was stirred at −78° C. during30 minutes and then, transferred into another round-bottom flaskcontaining L- or D-alanine isopropyl ethyl ester hydrochloride (14.91mmol) in DCM (30 mL) at 0° C. To the mixture was added TEA (31.31 mmol)over a period of 15 minutes. The reaction mixture was stirred at 0° C.during 1 hour and then, the solvent was evaporated. The residue wastriturated with ethyl acetate (45 mL) and the white solid wasfiltered-off. The filtrate was concentrated under reduced pressure andthe residue was purified by chromatography on silica gel (eluent:petroleum ether-petroleum ether/ethyl acetate 20%) to give the expectedintermediate.

Isopropyl (2S)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate(A22a)

60% yield; ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 1.15 (d, J=6.26 Hz, 3H), 1.16(d, J=6.26 Hz, 3H), 1.33 (m, 3H), 3.83 (dd, J=9.7 and 11.76 Hz, 1H),3.97-4.08 (m, 1H), 4.94 (heptuplet, J=6.26 Hz, 1H), 7.11-7.19 (m, 3H),7.27-7.35 (m, 4H), 8.16 (dd, J=1.72 and 9.07 Hz, 2H); ³¹P NMR (CDCl₃,161.98 MHz): δ (ppm) −3.21 (s, 0.45P), −3.18 (s, 0.55P); MS (ESI)m/z=409.14 (MH⁺).

Isopropyl (2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate(A22b)

80% yield; ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 1.22 (d, J=6.28 Hz, 3H), 1.23(d, J=6.28 Hz, 3H), 1.40 (m, 3H), 3.91-3.96 (m, 1H), 4.05-4.13 (m, 1H),5.01 (heptuplet, J=6.30 Hz, 1H), 7.19-7.25 (m, 3H), 7.33-7.41 (m, 4H),8.22 (dd, J=1.74 Hz and 8.95 Hz, 2H); ³¹P NMR (CDCl₃, 161.98 MHz): δ(ppm) −3.21 (s, 0.45P), −3.18 (s, 0.55P); MS (ESI) m/z=409.14 (MH⁺).

Butyl (2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate(A22c)

72% yield; yellow oil; ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 0.92 (t, J=7.35Hz, 3H), 1.30-1.39 (m, 2H), 1.40-1.43 (m, 3H), 1.56-1.63 (m, 2H),3.84-3.89 (m, 1H), 4.08-4.18 (m, 3H), 7.18-7.26 (m, 3H), 7.33-7.41 (m,4H), 8.23 (dd, J=1.77 Hz and 9.01 Hz, 2H); MS (ESI) m/z=423 (MH⁺).

Benzyl (2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate(A22d)

89% yield; yellow oil; ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 1.41-1.44 (m,3H), 3.82-3.88 (m, 1H), 4.13-4.25 (m, 1H), 5.14-5.15 (m, 2H), 7.18-7.24(m, 3H), 7.28-7.38 (m, 9H), 8.16-8.21 (m, 2H); MS (ESI) m/z=457 (MH⁺).

General Method F

The following procedure was used to obtain compounds 40i and 40ii.

To a solution of compound 301 (15 mmol) in THF (5 mL/mmol) was addedtert-butylmagnesium chloride (1M in THF) (31 mmol) over a period of 10minutes. Appropriate intermediate A22 (18 mmol) in THF (20 mL) was addedand the reaction mixture was stirred at room temperature during 3 days.The reaction mixture was quenched with saturated aqueous solution ofammonium chloride. The residue was suspended in ethyl acetate and washedwith water. The organic layer was washed with aqueous sodium bicarbonateand brine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by chromatography on silica gel(eluent: DCM-DCM/MeOH 2%) to separate the diastereoisomers.

Compound 40ii (Diastereoisomer 2)

White solid; 13% yield; ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.24-1.26 (m,6H), 1.36 (d, J=7.04 Hz, 3H), 1.59 (s, 3H), 3.69-3.77 (m, 1H), 3.91-3.99(m, 2H), 4.17-4.19 (m, 1H), 4.43-4.59 (m, 2H), 5.01-5.06 (m, 1H), 5.68(d, J=8.20 Hz, 1H), 6.42 (s, 1H), 7.21-7.39 (m, 5H), 7.60 (d, J=8.20 Hz,1H), 8.14 (s, 1H); ³¹P NMR (CDCl₃, 161.98 MHz): δ (ppm) 3.47 (s, 1P); MS(ESI) m/z=546.2 (MH⁺).

Compound 40i (Diastereoisomer 1)

In this case, after chromatography on silica gel, the mixture ofdiastereoisomers was purified by preparative HPLC.

White solid; 3% yield; ¹H NMR (CDCl₃, 400 MHz) δ 1.25 (d, J=6.25 Hz,6H), 1.38 (d, J=7.04 Hz, 3H), 1.51 (s, 3H), 3.66-3.74 (m, 2H), 3.82-3.96(m, 2H), 4.15 (dd, J=1.62 and 9.24 Hz, 1H), 4.39-4.53 (m, 2H), 5.03(heptuplet, J=6.26 Hz, 1H), 5.56 (dd, J=2.29 and 8.18 Hz, 1H), 6.39 (s,1H), 7.19-7.26 (m, 3H), 7.34-7.43 (m, 3H), 8.06 (s, 1H); ³¹P NMR (CDCl₃,161.98 MHz): δ (ppm) 3.35 (s, 1P); MS (ESI) m/z=546.20 (MH⁺).

The following abbreviations are used in Scheme 8:

General Method K

The following procedure was used to obtain compounds 202i and 205i.

To as solution of compound 301 (0.72 mmol) in anhydrous THF (7 mL/mmol)under nitrogen at room temperature was added tert-butylmagnesiumchloride (1M in THF) (1.52 mmol) followed by compound A22c or A22d(0.795 mmol) solubilized in THF (4 mL/mmol). DMSO (4 mL/mmol) was addedand the mixture was stirred at room temperature overnight. The reactionmixture was diluted with dichloromethane and washed with H₂O. Theorganic phase was dried, filtered and concentrated under reducedpressure. The residue was purified by chromatography on silica gel(eluent: DCM/MeOH 0 to 2%) followed by purification by preparative HPLCto give the expected pure diastereoisomers.

Compound 202i (Mixture of Diastereoisomers)

202i P-Diastereoisomer 1:

15% yield; white solid; ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 0.93 (t, J=7.37Hz, 3H), 1.32-1.40 (m, 2H), 1.40 (d, J=7.04 Hz, 3H), 1.51 (s, 3H),1.56-1.65 (m, 2H), 3.63 (d, J=7.70 Hz, 1H), 3.70-3.75 (m, 1H), 3.82-3.86(m, 1H), 3.92-4.02 (m, 1H), 4.08-4.19 (m, 3H), 4.39-4.52 (m, 2H), 5.56(d, J=8.20 Hz, 1H), 6.39 (s, 1H), 7.19-7.26 (m, 3H), 7.34-7.38 (m, 2H),7.41 (d, J=8.21 Hz, 1H), 8.10 (s, 1H); ³¹P NMR (CDCl₃, 161.98 MHz): δ(ppm) 4.27 (s, 1P); MS (ESI, El⁺) m/z=560 (MH⁺).

202i P-Diastereoisomer 2:

18% yield; white solid; ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 0.92 (t, J=7.35Hz, 3H), 1.30-1.38 (m, 2H), 1.37 (d, J=7.13 Hz, 3H), 1.57-1.62 (m, 2H),1.61 (s, 3H), 3.45-3.53 (m, 2H), 4.00-4.20 (m, 5H), 4.46-4.59 (m, 2H),5.63 (d, J=8.26 Hz, 1H), 6.44 (s, 1H), 7.19-7.22 (m, 3H), 7.34-7.38 (m,2H), 7.66 (d, J=8.18 Hz, 1H), 8.04 (s, 1H); ³¹P NMR (CDCl₃, 161.98 MHz):δ (ppm) 3.84 (s, 1P); MS (ESI, El⁺) m/z=560 (MH⁺).

Compound 205i (Mixture of Diastereoisomers)

205i P-Diastereoisomer 1:

12% yield; white solid; ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.40 (d, J=7.08Hz, 3H), 1.49 (s, 3H), 3.55 (d, J=7.83 Hz, 1H), 3.65-3.70 (m, 1H),3.77-3.81 (m, 1H), 3.97-4.04 (m, 1H), 4.07-4.10 (m, 1H), 4.28-4.45 (m,2H), 5.16 (s, 2H), 5.54 (d, J=8.22 Hz, 1H), 6.36 (s, 1H), 7.18-7.23 (m,3H), 7.31-7.38 (m, 8H), 7.99 (s, 1H); ³¹P NMR (CDCl₃, 161.98 MHz): δ(ppm) 4.07 (s, 1P); MS (ESI, El⁺) m/z=594 (MH⁺).

205i P-Diastereoisomer 2:

19% yield; white solid; ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 1.38 (d, J=7.11Hz, 3H), 1.59 (s, 3H), 3.41 (d, J=7.94 Hz, 1H), 3.51-3.56 (m, 1H),3.98-4.02 (m, 1H), 4.09-4.19 (m, 2H), 4.43-4.57 (m, 2H), 5.13 (s, 2H),5.61 (d, J=8.27 Hz, 1H), 6.43 (s, 1H), 7.18-7.22 (m, 3H), 7.28-7.39 (m,7H), 7.63 (d, J=8.16 Hz, 1H), 8.16 (s, 1H); ³¹P NMR (CDCl₃, 161.98 MHz):δ (ppm) 3.69 (s, 1P); MS (ESI, El⁺) m/z=594 (MH⁺).

Example 1B Preparation of Bridged Nucleosides

Isomer separation was carried out using a preparative SFC systemequipped with an AS-H chiral column and using methanol/CO₂ as the mobilephase. Synthesis of single diastereomers was performed as provided inScheme 2.

DMSO (163.7 mL, 2.31 mol) was added drop-wise to a solution of oxalylchloride (97.5 mL, 1.15 mol) in DCM (1.5 L) at −78° C. After 15 min atthis temperature a solution of A1 (200 g, 0.77 mol) in DCM (500 mL) wasadded drop-wise. After additional 15 min at −78° C. triethylamine (536mL, 3.84 mol, 5 eq) was added drop-wise. The reaction mixture wasallowed to warm to −20° C. then ethanol (1 L) and water (0.5 L) wereadded followed by portion-wise addition of NaBH4 (30.2 g, 0.8 mol, 1.04eq). The reaction mixture was allowed to warm to room temperature andstirred for 18 hrs. The reaction mixture was poured into 1M HCl aqueoussolution and extracted with DCM. The organic layers were washed withwater, brine, dried (MgSO4) and evaporated to give A2 (200 g, 100%) asan off-white solid. ¹H NMR (300 MHz, CDCl3) δ 5.80 (d, 1H), 4.60 (dd,1H), 4.30 (dt, 1H), 4.10-3.99 (m, 3H), 3.80 (dd, 1H), 2.57 (d, 1H), 1.56(s, 3H), 1.45 (s, 3H), 1.39 (s, 3H), 1.38 (s, 3H).

Preparation of Compound A3

NaH (60% in mineral oil, 14.4 g, 0.36 mol) was suspended in acetonitrile(600 mL) and cooled to 0° C. A solution of A2 (78.0 g, 0.3 mol) inacetonitrile (600 mL) was added drop-wise followed by a solution ofbenzyl bromide (42.8 mL, 0.36 mol) in acetonitrile (100 mL). Thereaction mixture was stirred for 4 h before careful addition of methanol(100 mL). The reaction mixture was partitioned between EtOAc and water.The aqueous layer was extracted with EtOAc. The organic layers werecombined and dried (MgSO4) and evaporated to give A3 (˜115 g, 100%) as awhite solid. ¹H NMR δ (300 MHz, CDCl3) δ 7.41-7.28 (m, 5H), 5.74 (d,1H), 4.77 (d, 1H), 4.59 (d, 1H), 4.57 (d, 1H), 4.37 (ddd, 1H), 4.13 (dd,1H), 4.04-3.92 (m, 2H), 3.88 (dd, 1H), 1.58 (s, 3H), 1.36 (s, 3H), 1.35(s, 3H).

Preparation of Compound A4

Acetic acid in water (80%, 1 L) was added to A3 (100 g, 0.29 mol) andthe mixture stirred at r.t. for 42 h. The reaction mixture was pouredinto a solution of NaOH solution (540 g in 3 L water) with vigorousstirring then extracted with EtOAc (×3). The organic layers werecombined and dried (MgSO4) then evaporated to give A4 (86.7 g, 98%) asyellow oil. ¹H NMR δ (300 MHz, CDCl3) δ 7.47-7.28 (m, 5H), 5.77 (d, 1H),4.78 (d, 1H), 4.65-4.50 (m, 2H), 4.16-4.10 (m, 1H), 4.03-3.97 (m, 1H),3.92 (dd, 1H), 3.75-3.61 (m, 2H), 2.50-2.38 (m, 2H), 1.59 (s, 3H), 1.36(s, 3H).

Preparation of Compound A5

A solution of A4 (23.66 g, 76.2 mmol) in water (250 mL) was slowly addedto a solution of sodium periodate (19.08 g, 89.2 mmol) in water (125 mL)at 0° C. After 30 min., ethylene glycol (2.5 mL) was added and thereaction mixture extracted with EtOAc. The organic layers were combined,dried (MgSO4) and evaporated to give A5 (20.44 g, 96%) as yellow oil. ¹HNMR δ (300 MHz, CDCl3) δ 9.61 (d, 1H), 7.42-7.24 (m, 5H), 5.81 (d, 1H),4.75 (d, 1H), 4.63 (d, 1H), 4.60 (t, 1H), 4.49 (dd, 1H), 3.85 (dd, 1H),1.60 (s, 3H), 1.36 (s, 3H).

Preparation of Compound A6

Aqueous 37% formaldehyde (40 mL) followed by 1N NaOH (200 mL) were addedto a solution of A5 (20.44 g, 73.45 mmol) in water (150 mL) and dioxane(50 mL) at 0° C. The reaction mixture was stirred at r.t. for 7 days andthen partitioned between EtOAc and brine. The organic layers werecombined, dried (MgSO4) and evaporated to give A6 (22.79 g, 100%) as apale yellow oil. ¹H NMR δ (400 MHz, CDCl3) δ 7.41-7.28 (m, 5H), 5.76 (d,1H), 4.80 (d, 1H), 4.62 (dd, 1H), 4.52 (d, 1H), 4.21 (d, 1H), 3.90 (dd,2H), 3.78 (dd, 1H), 3.55 (dd, 1H), 2.37 (t, 1H), 1.89 (dd, 1H), 1.63 (s,3H), 1.33 (s, 3H).

Preparation of Compound A7

A solution of A6 (84.98 g, 273.83 mmol) in pyridine (360 mL) was cooledto 0° C. and MsCl (63.89 mL, 825.47 mmol) was added portion wise. Afteraddition, the reaction slurry was stirred at room temperature for 3 hrsbefore cooled back to 14° C. 55 mL water was added drop-wise over 23 minand temperature rose up to 53° C. Then pyridine was mostly removed usingrotary evaporator (bath temperature <40° C.). The mixture was thenpartitioned into EtOAc 550 mL and water 500 mL. Organic layer wasfurther washed with brine and the first aqueous layer was also backextracted with EtOAc (200 mL). Organic layers were combined and driedover Na2SO4, evaporated and chased with acetonitrile (100 mL×2) and gaveyellow solid A7 (136.61 g, 91.83%). ¹H NMR δ (400 MHz, CDCl3) δ7.42-7.28 (m, 5H), 5.80 (d, 1H), 4.90 (d, 1H), 4.80 (d, 1H), 4.67 (m,1H), 4.60 (d, 1H), 4.33 (2, 1H), 4.20 (d, 1H), 4.16 (d, 1H), 3.10 (s,3H), 3.00 (s, 3H), 1.70 (s, 3H), 1.36 (s, 3H) LCMS [M] 485.2.

Preparation of Compound A8

Compound A7 (136.61 g, 268.9 mmol) was slowly dissolved into acetic acid(1.25 L) and the solution was cooled to 7° C. before acetic anhydride(190 mL, 2010 mmol, 7.5 eq) and concentrated H2SO4 (1.72 mL, 33 mmol,0.12 eq) were added. The solution was stirred for another 10 min. beforewarmed up to room temperature. The solution was stirred at roomtemperature for 18 hrs and then cooled to 9° C. 120 mL of water wasadded over 2 min and the mixture was stirred at room temperature for 1hr, before it was partitioned between water 964 mL and DCM 1140 mL.Organic layer was isolated and evaporated in order to remove acetic acidand gave yellow oil. The oil was re-dissolved into DCM (820 mL) andwashed twice with saturated NaHCO3 solution. The solution was dried overNa2SO4 and evaporated to give yellow oil A8 (127.3 g, 94.7%). ¹H NMR δ(400 MHz, CDCl3) δ 7.41-7.29 (m, 5H), 6.20 (S, 1H), 5.40 (d, 1H), 4.64(d, 1H), 4.52 (m, 2H), 4.44 (m, 1H), 4.39 (d, 1H), 4.31 (d, 1H), 4.21(m, 1H). LCMS [M+CH3COO—] 569.0.

Preparation of Compound A9

At room temperature, into the mixture of A8 (113.04 g, 221.4 mmol) andchloropurine (41.31 g, 243.6 mmol) in 1,2-dichloroethane (1.36 L), wasadded N,O-Bis(trimethylsilyl)acetamide (108.4 mL, 443.3 mmol). Theslurry was then heated to 81° C. for 40 min. and cooled back to 29° C.TMSOTf (81.34 mL, 444.9 mmol) was added all at once and temperature roseto 36° C. The mixture was then heated to 81° C. again for 2 hrs beforeit was cooled down to room temperature. 1,2-dichloroethane was thenremoved using rotary evaporator and remaining mixture was partitionedinto DCM (1.15 L) and saturated NaHCO3 solution (0.64 L). Solid crashedand slurry was filtered and solid rinsed with 65 mL of DCM. Filtrate andrinse were combined and the organic layer was washed again with satNaHCO3 solution, 5% brine solution and dried over Na2SO4, evaporated togive a foamy solid A9 (136.79 g, 94.7%). ¹H NMR δ (400 MHz, CDCl3) δ7.69 (S, 1H), 7.34-7.27 (m, 5H), 5.92 (d, 1H), 5.57 (t, 1H), 5.32 (b,2H), 5.13 (d, 1H), 4.73 (d, 1H), 4.60 (m, 3H), 4.33 (t, 2H), 2.97 (s,3H), 2.94 (s, 3H), 2.04 (s, 3H) LCMS [M] 620.15

Preparation of Compound A10

Compound A9 (136.79 g, 207.4 mmol) was mixed with 1.3 L THF and 1.3 LEtOH. The solution was cooled to 0° C., before NaOEt (95%, 81.71 g,1140.6 mmol) was added in portions. The mixture was stirred and warmedup to room temperature over 18 hrs. The mixture was then cooled to 0° C.before HCl 2N (650 mL) was added in portions. Organic solvents wereremoved and remaining crude oil was partitioned into 1.0 L EtOAc and 150mL water. Aqueous layer was back extracted with EtOAc (200 mL) and allorganic layers were combined and washed with 5% brine solution (400mL×4). Organic layer was isolated and dried over Na2SO4, evaporated andgave brown powder A10 (100.0 g, 93.9%). ¹H NMR δ (400 MHz, CDCl3) δ 7.63(S, 1H), 7.37-7.30 (m, 5H), 5.93 (d, 1H), 4.93 (b, 2H), 4.76 (S, 1H),4.73-4.54 (m, 6H), 4.40 (S, 1H), 4.20 (d, 1H), 4.01 (d, 1H), 3.04 (S,3H), 1.49 (t, 3H). LCMS [M+H] 492.19.

Preparation of Compound A11

Compound A10 (113.5 g, 219.7 mmol) was dissolved into DMSO (114 mL).Then NaOBz powder (99.41 g, 689.9 mmol) was added. The slurry was heatedto 97° C. for 2.5 hrs before it was cooled down and mixed with water (1L), and then with EtOAc (1 L). Aqueous layer was further washed withEtOAc (0.7 L) and all EtOAc layers were combined and washed withsaturated NaHCO3 solution (0.72 L), and with 5% brine solution (0.75L×2). EtOAc layer was dried over Na2SO4, evaporated and gave powder A11(118.6 g, 93.6%). ¹H NMR δ (400 MHz, CDCl3) δ 7.93 (t, 2H), 7.87 (s,1H), 7.69 (m, 1H), 7.53 (m, 2H), 7.34-7.28 (m, 5H), 6.54 (b, 2H), 5.92(S, 1H), 4.82 (S, 1H), 4.76 (d, 2H), 4.71 (d, 2H), 4.59 (s, 1H), 4.46(m, 2H), 4.12 (m, 1H), 4.05 (m, 1H), 1.36 (t, 3H). LCMS [M+H] 518.27.

Preparation of Compound A12

Compound A11 (118.6 g, 214.5 mmol) was dissolved into THF (1.1 L). Theninto the solution was added NaOH aq (NaOH 30.89 g, 772.2 mmol, 3.6 eq,with water 0.5 L) at room temperature. The mixture was stirred over 16hrs and then heated to 35° C. for 6.5 hrs. The reaction mixture wascooled to 1° C. and HCl (1N 550 mL) was added. Organic layer and aqueouslayer were separated. The aqueous layer was back extracted with EtOAc(0.5 L) and both organic layers were combined and washed with saturatedNaHCO3 (450 mL) and then with 5% brine (450 mL×2). Brine washes werecombined and washed with EtOAc (200 mL). All organic layers werecombined and dried over Na2SO4, evaporated and gave crude solid (103 g).The crude solid was purified on column (1.5 kg Gold combiflash column,with solvents DCM and EtOAc), and gave pure solid compound A12 (56.49 g,96%). ¹H NMR δ (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.34-7.27 (m, 5H), 6.53(br, 2H), 5.84 (s, 1H), 5.15 (t, 1H), 4.65 (d, 3H), 4.46 (q, 2H), 4.29(s, 1H), 3.95 (d, 1H), 3.81 (m, 3H), 3.18 (d, 1H), 1.36 (t, 3H). LCMS[M+H] 414.20.

Preparation of Compound A15

To a stirred solution of D-alanine isopropyl ester HCl A13 (14.2 g,84.66 mmol) and phenyl dichlorophosphate 14 (12.6 mL, 84.66 mmol) in DCM(142 mL) at −70° C. was added a solution of triethylamine (24.7 mL) inDCM (142 mL) over 50 min. The mixture was stirred at this temperaturefor additional 1.5 hrs. The mixture was filtered through a sinteredglass funnel and the filtrate was concentrated under reduced pressure.The residue was triturated with TBME (120 mL), filtered off and rinsedwith TBME (2×120 mL). The combined filtrate was concentrated underreduced pressure to give A15 (25.9 g, 100%), which was used for thefollowing coupling reaction without further purification.

Preparation of Compound A16

To a stirred solution of nucleoside A12 (10 g, 24.19 mmol) andN-methylimidazole (15.4 mL, 193.52 mmol) in DCM (200 mL) at 5° C., wasadded a solution of compound A15 (25.9 g, 84.66 mmol) in DCM (45 mL)over 1 hr. The mixture was allowed to warm to rt overnight and thenconcentrated under reduced pressure to give yellow oil. This oil wasdiluted with EtOAc (200 mL) and water (200 mL). The organic layer wasseparated, washed with 5% aqueous ammonium chloride solution (2×200 mL)and 5% brine solution (200 mL), dried (sodium sulphate), filtered andconcentrated under reduced pressure to give crude product (29.9 g). Thecrude compound was chromatographed using EtOAc/dichloromethane 3:2gradient to give product A16 (13.8 g, 83%) as off-white solid. ¹H NMR(DMSO-d6) δ 7.92 (s, 1H), 7.17-7.34 (m, 10H), 6.54 (br s, 2H), 6.07 (q,1H), 5.88 (d, 1H), 4.84 (m, 1H), 4.76 (d, 1H), 4.68 (d, 1H), 4.47 (m,5H), 4.03 (m, 1H), 3.84 (m, 2H), 1.37 (t, 3H), 1.19 (m, 4H), 1.13 (m,6H); 31P NMR 3.70, 3.48; HPLC (test20) 5.42 min; LCMS 16.35 min (M++H)683.33.

Preparation of 425 Isomer Mixture

To a stirred mixture of Pd/C (5.4 g) in EtOH (140 mL) at 22° C. wasadded a solution of nucleoside A16 (13.8 g, 20.21 mmol) in EtOH (560mL), and the reaction mixture was heated to 50° C. for 45 min. The crudemixture was filtered through a Celite pad and rinsed with MeOH (4×250mL). The combined filtrate was concentrated under reduced pressure togive 12.4 g of crude product. The crude compound was chromatographedusing 0-5% MeOH/dichloromethane gradient to give 425 (mixture ofisomers, 9.3 g, 77% yield) as an off-white solid. ¹H NMR (DMSO-d6) δ7.96 (s, 1H), 7.17-7.37 (m, 5H), 6.54 (br s, 2H), 6.06 (q, 1H), 5.90 (d,1H), 5.81 (d, 1H), 4.86 (m, 1H), 4.32-4.47 (m, 6H), 3.97 (d, 1H), 3.79(m, 2H), 1.37 (t, 3H), 1.34 (m, 3H), 1.16 (m, 6H); 31P NMR 3.83, 3.63;HPLC (test20) 4.34 min; LCMS 11.72 min (M++H) 593.27.

Preparation of the Compound A17

To a stirred solution of D-alanine isopropyl ester hydrochloride A13 (20g, 119.3 mmol) and phenyl dichlorophosphate (25.3 g, 17.9 mL, 118.8mmol) in anhydrous dichloromethane (150 mL) was added a solution oftriethylamine (25.4 g, 35 mL, 251.3 mmol) in anhydrous dichloromethane(150 mL) at −70° C. over 45 min dropwise. The reaction mixture wasstirred at this temperature for additional 30 min and then allowed towarm to 0° C. over 2 h and stirred for 1 h. To this mixture was added asolution of 2,3,4,5,6-pentafluoro phenol (22 g, 119.5 mm01) andtriethylamine (1.3 g, 17 mL, 122 mmol) in anhydrous dichloromethane (75mL) over 40 min. The crude mixture was stirred at 0° C. for 2 h and thenstored at 5° C. over night. The white solid (triethylaminehydrochloride) was filtered off and washed with dichloromethane (1×25mL). The filtrate was concentrated under reduced pressure, the residuewas triturated with TBME (300 mL) and the triethylamine hydrochloridesalt was removed by filtration. The cake was washed with dichloromethane(2×25 mL) and the combined filtrate was concentrated under reducedpressure to give the crude solid containing even mixture ofdiastereomers. The mixture was triturated with 20% EtOAc in hexanes (200mL) to give 29.5 g of compound A17 as a white solid. This was furtherpurified using a mixture of IPA (240 mL) and water (290 mL) to give thedesired compound A17 (21.5 g, 40%). ³¹P NMR (CDCl3, 162 MHz) δ −1.56; ¹HNMR (CDCl3, 400 MHz) δ 7.40-7.36 (m, 2H), 7.29-7.21 (m, 3H),5.10-5.011H), 4.21-4.02 (m, 2H), 1.47 (d, J=7.2 Hz, 3H), 1.29-1.24 (m,6H).

Preparation of Compound A18

To the stirred solution of compound A17 (1.5 g, 3.63 mmol) in dry THF(35 mL) was added a 1.0 M solution of tert-butylmagnesium chloride inTHF (4.5 mL, 5.4 mmol) over 7 min at −9° C. The reaction mixture wasstirred at that temperature for 10 min and a solution of compound A2 (2g, 4.4 mmol) in THF (10 mL) was added over 10 min at −9° C. The crudereaction mixture was stirred at that for additional 40 min, warmed to rtover a period of 1 h, and then quenched with 2 N HCl (20 mL). Toluene(100 mL) was added and the layers separated, aqueous layer re-extractedwith toluene (50 mL). The combined toluene layer was washed with brine(1×50 mL), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to give the crude product (4.2 g).The crude compound was chromatographed using 0-5% MeOH/dichloromethanegradient to give product A18 (2.2 g, yield—88%). ³¹P NMR (CDCl3, 162MHz) δ 2.43; ¹H NMR (CDCl3, 400 MHz) δ 7.68 (s, 1H), 7.29-7.22 (m, 9H),7.16-7.12 (m, 1H), 5.90 (s, 1H), 5.02-4.98 (m, 2H), 4.64-4.55 (m, 2H),4.46-4.45 (m. 3H), 4.13-4.11 (m, 2H), 4.02-3.92 (m, 2H), 3.83-3.80 (m,1H), 1.48 (t, J=7.2 Hz, 3H), 1.39-1.37 (m, 4H), 1.27-1.19 (m, 6H); LCMS:683.33 (MH+).

Preparation of 425

To a stirred solution of compound A18 (2.0 g, 2.93 mmol) in ethanol (40mL) was added Pd/C (10%, 1.1 g). The crude mixture was heated at 50° C.and ammonium formate (0.96 g, 15.24 mmol) was added. The reactionmixture was heated for additional 1.5 hrs and filtered over a pad ofcelite. The celite bed washed with MeOH (30 mL) and the filtrate wasconcentrated to give 3 g of crude product. The crude product waschromatographed using 0-5% MeOH/dichloromethane gradient to give 25 (1.0g, yield 58%). ³¹P NMR (CDCl3, 162 MHz) δ 3.61; ¹H NMR (DMSO-d6, 400MHz) δ 7.93 (s, 1H), 7.38-7.34 (m, 2H), 7.23-7.15 (m, 3H), 6.15 (bs,2H), 6.09-6.04 (m, 1H), 5.95 (d, J=4 Hz, 1H), 5.80 (s, 1H), 4.89-4.86(m, 1H), 4.51-4.29 (m, 6H), 3.99 (d, J=8 Hz, 1H), 3.82-3.73 (m, 2H),1.35 (t, J=7.2 Hz, 3H), 1.23 (d, J=7.2 Hz, 3H), 1.16-1.14 (m, 6H); LCMS:593.23 (MH+).

Preparation of 401

(2S)-isopropyl2-(((((1R,3R,4R,7S)-3-(2-amino-6-oxo-1H-purin-9(6H)-yl)-7-(benzyloxy)-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (B6)

To a solution of phenyl dichlorophosphate (739 μL, 4.7 mmol) in THF (10mL) at −55.6° C. under Argon, was added L-alanine isopropyl ester (827mg, 4.93 mmol, 1.05 eq.) dissolved in 8.3 mL of DCM over 5 min. (−44.3°C.), Triethylamine (1.38 mL, 9.87 mmol, 2.1 eq.) was added over 3 min(−48.6° C.→−40° C.). The reaction was kept <−30° C. and reactionfollowed by LCMS, ¹H and ³¹P NMR that indicated reaction completionafter 25 min. to give compound B4.

To a suspension of B5 (1 g, 2.35 mmol, 0.5 eq.) in THF/DCM (10/5 mL) at−40° C. under Argon, was added t-BuMgCl (5.17 mL, 5.17 mmol, 1.1 eq.)over 4 min. (−32.1° C.). The reaction mixture was kept stirring at 0° C.for 45 min (B5 completely solubilized). To this solution cooled at −50°C. was added the previous chlorophosphoramidate solution (compound 4)over 7 min (−36.8° C.) and 5 mL of THF was used to rinse the remainingphosphoramidate compound. The reaction was kept stirring at 0° C. for 30min., the reaction was followed by LCMS.

To the reaction mixture was added 25 mL 5% brine and 25 mL ethylacetate. The organic was separated then washed with 20 mL 5% brine. Theorganic layer was then dried over Na₂SO₄ and concentrated to give 2.47 gof a yellow oil. The crude product was purified by Combiflash (8O g goldcolumn, DCM 100%→DCM/MeOH 90/10). Compound B6 was isolated as a whitesolid (818.8 mg, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (d, J=4 Hz, 1H),7.36-7.30 (m, 7H), 7.19-7.15 (m, 3H), 6.54 (br s, 2H), 6.08 (m, 1H),5.88 (d, J=8 Hz, 1H), 5.01 (m, 1H), 4.77 (d, J=12 Hz, 1H), 4.66 (m, 2H),4.50-4.38 (m, 5H), 4.02 (dd, J=8 Hz, 20 Hz, 1H), 3.86-3.74 (m, 2H), 1.39(t, J=8 Hz, 3H), 1.23 (m, 9H). ³¹P NMR (400 MHz, DMSO-d₆) δ 3.72, 3.65.(M+H⁺) 709.

(2S)-isopropyl2-(((((1R,3R,4R,7S)-3-(2-amino-6-oxo-1H-purin-9(6H)-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-1-yl)methoxy)(phenoxy)phosphoryl)amino)proapnoate(401)

To a solution of B6 (350 mg, 0.484 mmol) in ethanol (17.5 mL) was addedPd/C (128.7 mg, 0.121 mmol, 0.25 eq.). The mixture was heated at 50° C.and ammonium formate (152.6 mg, 2.42 mmol, 5 eq.) was added. Thereaction mixture was kept stirring at 50° C. for 30 min. After coolingdown to room temperature, the mixture was filtered through a celite padand solid rinsed with 3×10 mL of methanol. The filtrate was concentratedand dissolved in 10 mL DCM, washed with 10 mL ¼ saturated. NaHCO₃solution, 10 mL water. The second organic solution was extracted with 10mL DCM. The combined organic phases were dried over Na₂SO₄ andconcentrated to give 311 mg of a white solid. This crude product waspurified using Combiflash (4 g gold column DCM 100%→DCM/MeOH 90/10).Compound 7 was isolated as a white solid (237 mg, 79%). ¹H NMR (400 MHz,DMSO-d₆) δ 7.95 (s, 1H), 7.37-7.35 (m, 2H), 7.22-7.18 (m, 3H), 6.54 (brs, 2H), 6.04 (m, 1H), 5.90-5.76 (m, 2H), 4.87 (sept, J=4 Hz, 1H),4.49-4.4 (m, 5H), 4.30 (t, J=4 Hz, 1H), 4.01 (dd, J=8 Hz, 24 Hz, 1H),3.85 (m, 2H), 1.38 (t, J=8 Hz, 3H), 1.25 (m, 3H), 1.16 (m, 6H). ³¹P NMR(400 MHz, DMSO-d₆) δ 3.85, 3.73. (M+H⁺).

Example 1C Compound 502a

To a stirred solution of 4-nitrophenyl dichlorophosphate (Aldrich)(35.97 mmol) in DCM (2 mL/mmol) was added a solution of phenol (Aldrich)(35.97 mmol) and TEA (39.57 mmol) in DCM (2 mL/mmol) at −78° C. over aperiod of 20 minutes. The reaction mixture was stirred at −78° C. during30 minutes and then, transferred into another round-bottom flaskcontaining D-alanine isopropyl ester hydrochloride (35.97 mmol) in DCM(2 mL/mmol) at 0° C. To the mixture was added TEA (31.31 mmol) over aperiod of 15 minutes. The reaction mixture was stirred at 0° C. during 1hour and then, the solvent was evaporated. The residue was trituratedwith ethyl acetate (45 mL) and the white solid was filtered-off. Thefiltrate was concentrated under reduced pressure and the residue waspurified by chromatography on silica gel (eluent: petroleumether-petroleum ether/ethyl acetate 20%) to give the expected compoundin 80% yield; ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 1.22 (d, J=6.28 Hz, 3H),1.23 (d, J=6.28 Hz, 3H), 1.40 (m, 3H), 3.91-3.96 (m, 1H), 4.05-4.13 (m,1H), 5.01 (heptuplet, J=6.30 Hz, 1H), 7.19-7.25 (m, 3H), 7.33-7.41 (m,4H), 8.22 (dd, J=1.74 and 8.95 Hz, 2H); ³¹P NMR (CDCl₃, 161.98 MHz): δ(ppm) −3.21 (s, 0.45P), −3.18 (s, 0.55P); MS (ESI) m/z=409.14 (MH⁺).

Compound 502a

To a solution of 3′-deoxy nucleoside (0.803 mmol) in anhydrous THF (4mL) at room temperature under nitrogen was added dropwisetert-butylmagnesium chloride (1M in THF) (1.69 mmol) followed by DMSO(0.6 mL). The heterogeneous reaction mixture was stirred during 30minutes at room temperature. Compound B2 (0.964 mmol) in THF (2.4 mL)was added dropwise and the reaction mixture was abandoned at roomtemperature all the weekend. The reaction mixture was quenched withsaturated aqueous solution of NH₄Cl and diluted with ethyl acetate. Themixture was extracted with ethyl acetate and the organic layer waswashed with H₂O and NaHCO₃. The organic layer was dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by chromatography on silica gel (eluent: CH₂Cl₂—CH₂Cl₂/CH₃OH)and by preparative HPLC to give two pure diastereoisomers.

Compound 502a, Diastereoisomer 1:

white solid; 14% yield; ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm) 1.09 (d,J=6.24 Hz, 3H), 1.12 (d, J=6.24 Hz, 3H), 1.13 (d, J=21.79 Hz, 3H), 1.19(d, J=7.11 Hz, 3H), 1.35 (t, J=7.11 Hz, 3H), 2.28-2.37 (m, 1H),3.70-3.80 (m, 1H), 4.23-4.29 (m, 1H), 4.35-4.40 (m, 1H), 4.45 (q, J=7.11Hz, 2H), 4.47-4.51 (m, 1H), 4.83 (heptuplet, J=6.24 Hz, 1H), 6.04-6.09(m, 1H), 6.06 (d, J=18.25 Hz, 1H), 6.56 (s, 2H), 7.14-7.17 (m, 1H),7.20-7.22 (m, 2H), 7.32-7.36 (m, 2H), 7.94 (s, 1H); ³¹P NMR (DMSO-d₆,161.98 MHz) δ (ppm) 3.6 (s, 1P); MS (ESI) m/z=581.12 (MH⁺).

Compound 502a, Diastereoisomer 2: white solid; 6% yield; ¹H NMR(DMSO-d₆, 400 MHz) δ (ppm) 1.10 (d, J=6.21 Hz, 3H), 1.11 (d, J=6.21 Hz,3H), 1.13 (d, J=6.95 Hz, 3H), 1.16 (d, J=21.98 Hz, 3H), 1.35 (t, J=7.10Hz, 3H), 2.28-2.37 (m, 1H), 3.70-3.80 (m, 1H), 4.26-4.32 (m, 1H),4.38-4.43 (m, 1H), 4.44 (q, J=7.12 Hz, 2H), 4.47-4.54 (m, 1H), 4.81(heptuplet, J=6.23 Hz, 1H), 5.98 (dd, J=9.96 Hz and 12.72 Hz, 1H), 6.09(d, J=18.27 Hz, 1H), 6.55 (s, 2H), 7.14-7.18 (m, 3H), 7.33-7.37 (m, 2H),7.99 (s, 1H); ³¹P NMR (DMSO-d₆, 161.98 MHz) δ (ppm) 3.97 (s, 1P); MS(ESI) m/z=581.08 (MH⁺).

Example 1D Preparation of 4′-fluoro nucleosides Compound 602b TwoDiastereomers

Uridine (10 grams, 40.1 mmol) was dissolved in acetone (100 mL)containing sulfuric acid (conc., 1.0 mL). After stirring at roomtemperature overnight, the mixture was concentrated under reducedpressure. The crude product was purified by flash column chromatography(Silica Gel, 100% DCM to 4% MeOH/DCM) to afford 11.0 grams of theacetonide A2 (98%).

HPLC (Method A, 254 nm) split peak at 2.3 and 2.49, 99.6 A %; ¹H NMR(400 MHz, CDCl₃) δ 1.34 (s, 3H), 1.56 (s, 3H), 2.50-2.60 (br-s, 1H),3.79-3.90 (m, 2H), 4.27 (m, 1H), 4.95 (m, 1H), 5.02 (m, 1H), 5.53 (d,1H), 5.71 (dd, 1H), 7.33 (d, 1H), 8.03 (br s, 1H).

Step 2:

The acetonide A2 (11.0 g, 38.7 mmol) was suspended in dichloromethane(110 mL). Dimethylaminopyridine (DMAP, 11.8 g, 96.8 mmol, 2.5 eq) wasadded and the mixture stirred at room temperature until the acetonidehad fully dissolved. The mixture was cooled to ca. 0° C. (ice-bath) andtosyl chloride (8.85 g, 46.4 mmol, 1.2 eq) was added in 5 portions.After the addition was complete, the ice bath was removed and themixture stirred for 1 hour. HPLC analysis showed the reaction to becomplete. The mixture was transferred to a reparatory funnel and waswashed with aqueous HCl (1N, 2×100 mL), aqueous sodium bicarbonate(saturated, 100 mL), and brine (100 mL). The organic solution was driedover magnesium sulfate and was concentrated under reduced pressureaffording the crude tosylate. (15.47 g, 91%). The crude product A3(purity; ca 86% by NMR) was used without purification for Step-3.

HPLC (Method A, 254 nm), 4.86 min; LCMS (m/e 327.05, M⁺-Uracil); ¹H NMR(400 MHz, CDCl₃) δ 1.29 (s, 3H), 1.50 (s, 3H), 2.40 (s, 3H), 4.22 (m,2H), 4.30 (m, 1H), 4.76 (dd, 1H), 4.90 (dd, 1H), 5.61 (d, 1H), 5.67 (d,1H), 7.21 (d, 1H), 7.29 (d, 2H), 7.72 (d, 2H), 9.39 (br s, 1H).

Step 3:

The crude tosylate A3 (39.5 g, 78.7 mmol) was dissolved in THF (100 mL)and was cooled to −10° C. Potassium t-butoxide (26.5 g, 236 mmol, 3 eq)was added forming a solid mass. An additional 250 mL of THF was added toensure adequate stirring. The mixture was stirred for 30 minutes andHPLC analysis showed that the reaction was complete. Silica gel (60 g)was added and the mixture was concentrated under reduced pressure. Thecrude product was purified by flash column chromatography (Silica Gel,100% DCM to 4% MeOH/DCM) to afford 13.2 g (62%) of the enol ether A4.

HPLC (Method A, 272 nm), 3.24 min, 98% A; LCMS (M⁺+1 m/e 267.09); ¹H NMR(400 MHz, CDCl₃) δ 1.41 (s, 3H); 1.53 (s, 3H), 4.42 (m, 1H), 4.60 (m.1H), 5.05 (m, 1H), 5.33 (m, 1H), 5.67 (s, 1H), 5.75 (dd, 1H), 7.20 (d,1H), 9.60 (br s, 1H).

Step 4:

The nucleosidic enol-ether A4 (7.34 g, 27.6 mmol, 1 eq) and finelycrushed silver fluoride (17.5 g, 138 mmol, 5 eq) were added to a flaskcontaining dichloromethane (520 mL, DCM was needed to ensure adequatestirring of the heterogeneous mixture.) The suspension was stirredrapidly and cooled to 0° C. In a separate flask, iodine (14.0 g, 55.2mmol, 2 eq) was dissolved in THF (40 mL). (The limited solubility ofiodine in DCM resulted in incomplete reaction when DCM was used forpreparing the iodine solution.) The iodine solution was transferred to aslow-addition funnel and was added to the reaction mixture over 70minutes. This addition rate provided a 7:1 ratio of the desired isomer(R) to undesired isomer (S). The mixture was stirred for 10 min at whichpoint HPLC analysis showed the reaction to be complete. The reactionmixture was quenched by the addition of an aqueous solution of NaS₂O₃and NaHCO₃ (5 wt % each, 300 mL total volume). The mixture was filteredthrough Celite™ and the filter pad washed with DCM. The biphasic mixturewas transferred to a reparatory funnel and the phases were separated.The organic phase was dried with magnesium sulfate and the mixtureconcentrated under reduced pressure affording ca. 11 g of crude product.The crude product was purified by flash column chromatography (SilicaGel, 0 to 60% EtOAc/heptane) to provide A5 as a beige colored solid. Thecrude solid was dissolved in DCM (20 mL) which was then added to heptane(200 mL) giving A5 as a white-colored solid. (A5, 10.4 g, 82%).

HPLC (Method A, 254 nm); A5 (4.18 and 4.38 min) 97% A, 7:1 R:S; ¹H NMR(400 MHz, CDCl₃) δ 9.16 (br s, 1H), 7.20 (d, 1H), 5.77 (d, 1H), 5.65 (s,1H), 5.16 (m, 1H), 5.10 (m, 1H), 3.53 (m, 1H), 3.48 (m, 1H), 1.59 (s,3H), 1.38 (s, 3H); ¹⁹F NMR (376 MHz, CDCl₃) δ −101.91 (1F, A5-R, Major),−94.16 (0.165F, Minor, A5-S).

Step 5:

The iodofluorinated nucleoside A5 (2.4 g, 5.8 mmol, 1 eq) was dissolvedin DMF (24 mL). Sodium azide (1.9 g, 29 mmol, 5 eq) was added and themixture stirred and heated at 100° C. overnight. HPLC analysis indicatedthat the reaction was incomplete. Additional sodium azide (378 mg, 5.8mmol, 1 eq) was added and the reaction continued for another 105minutes. HPLC analysis showed that the reaction was nearly complete. Themixture was allowed to cool to room temperature and ethyl acetate (75mL) and water (50 mL) were added. The mixture was then transferred to aseparatory funnel and the phases were split. The aqueous phase wasextracted with ethyl acetate (25 mL). The combined organic layers werewashed with water (4×50 mL), dried over magnesium sulfate, andconcentrated under reduced pressure. The crude product was purified byflash column chromatography (Silica Gel, 0 to 60% EtOAc/heptane) toprovide 1.63 g of the desired azide A6 (86%).

HPLC (Method A, 254 nm); A6, 3.96 min, 4.09 min; ¹H NMR (400 MHz, CDCl₃)δ 8.90 (br s, 1H), 7.18 (d, 1H), 5.77 (dd, 1H), 5.68 (s, 1H), 5.10 (m,2H), 3.57 (d, 1H), 3.54 (s, 1H), 1.60 (s, 3H), 1.38 (s, 3H); ¹⁹F NMR(376 MHz, CDCl₃) δ −109.70 (1F, A6-R, Major), −102.10 (0.280F, A6-S,Minor).

Step 6:

The azido nucleoside A6 (0.988 g, 3.2 mmol, 1 eq) was dissolved inacetonitrile (10 mL). The mixture was cooled to 0° C. (ice-bath) andnitrosyl tetrafluoroborate (1.06 g, 9.06 mmol, 3 eq) was added in asingle portion. The mixture was stirred for 30 minutes at 0° C. Theice-bath was removed and the mixture stirred for 1 hour at roomtemperature. HPLC analysis showed the reaction to be complete. Thereaction was quenched by the addition of 50% brine/50% Na₂HPO₄ (20 mL).The mixture was transferred to a separatory funnel and was extractedwith dichloromethane (3×20 mL). The combined organic extracts were driedwith magnesium sulfate and concentrated under reduced pressure affording0.699 g (81%) of crude A7. The crude material was used in Step 7 withoutfurther purification.

HPLC (Method A, 254 nm); A7, 2.77 min; LCMS (M⁺+1, m/e=285).

Step 7:

The nucleoside A7 (699 mg, 2.5 mmol, 1 eq) was dissolved in THF (6.3 mL)and water (0.7 mL). TFA (35 μL) was added and the mixture stirred for 1hour at room temperature. HPLC analysis showed that the reaction wascomplete. The mixture was concentrated under reduced pressure. The crudeproduct was purified by flash column chromatography (Silica Gel, 100%DCM to 4% MeOH/DCM) to provide 308 mg (41%) of the hydroxymethylnucleoside A8.

HPLC (Method A, 254 nm); A8, 2.74 min; LCMS (M⁻−1, m/e=301); ¹H NMR (400MHz, CDCl₃) δ 1.38 (s, 3H), 1.59 (s, 3H), 2.41 (br s, 1H), 3.82 (d, 2H),5.10 (d, 1H), 5.24 (m, 1H), 5.72 (s, 1H), 5.77 (d, 1H), 7.23 (d, 1H),9.06 (br s, 1H); ¹⁹F NMR (376 MHz, CDCl₃) δ −115.65.

Step 8a:

Phenyl dichlorophosphate (495 μL, 3.31 mmol, 1 eq) was dissolved in THF.The mixture was cooled to −66° C. In a separate flask, a solution ofisopropyl alanine (583 mg, 3.48 mmol, 1.05 eq) in DCM (6 mL) wasprepared. This solution was added to the solution of thedichlorophosphate over 5 minutes. Triethylamine (966 μL, 6.95 mmol, 2.1eq) was then added over 3 minutes maintaining the temperature at −66° C.The mixture was stirred for 25 minutes and this solution was used forStep 8 without further purification.

Step 8:

The nucleoside A8 (500 mg, 1.65 mmol, 0.5 eq) was dissolved in THF (5mL) forming a clean solution. The mixture was stirred and cooled to −43°C. t-Butyl magnesium chloride (1M in THF, 3.64 mL, 3.64 mmol, 1.1 eq)was added drop-wise over 5 minutes. The mixture was cooled to 50° C. andthe solution of the chlorophosphamidate A13 (3.31 mmol, 1 eq) was addeddrop-wise via a syringe over 7 minutes. (The solution becamebrown-colored and cloudy.) The mixture was stirred for 30 minutes andanalyzed by HPLC. The mixture was warmed to 0° C. and stirred for 30minutes. LCMS analysis indicated the reaction to be complete. Brine (5%,10 mL) was added, the mixture was transferred to a reparatory funnel andwas extracted with ethyl acetate (3×15 mL). The organic extracts weredried over magnesium sulfate and were concentrated under reducedpressure. The crude product was purified by flash column chromatography(Silica Gel, 100% DCM to 4% MeOH/DCM) to afford 324 mg (34%) of themixture of the phosphoramidate diastereomers A9.

HPLC (Method A, 254 nm); A9, 4.87 min, 4.95 min 1.8:1 ratio ofdiastereomers; LCMS (M⁻−1, m/e=570); ¹H NMR (400 MHz, CDCl₃) δ 1.18 (m,6H), 1.31 (m, 3H), 1.35 (m, 3H), 1.55 (s, 3H), 3.98 (m, 2H), 4.28 (m,2H), 4.98 (m, 2H), 5.20 (m, 1H), 5.69 (m, 1H), 5.78 (s, 1H), 7.20, 7.28(m, 6H), 9.22, 9.41 (2s, 1H); ¹⁹F NMR (376 MHz, CDCl₃) δ −113.99 (m,1F), −113.53 (m, 0.6F); ³¹P NMR (162 MHz, CDCl₃), 2.33, 2.32 (2s, 1P).

Step 9:

The nucleoside A9 (548 mg, 0.959 mmol, 1 eq) was dissolved in formicacid (80%, 35 mL). The mixture was stirred a room temperature for 3 hourand 45 minutes. HPLC analysis showed the reaction to be complete. Thereaction mixture was transferred to a reparatory funnel, was dilutedwith brine (35 mL) and was extracted with ethyl acetate (3×40 mL). Thecombined organic extracts were dried over magnesium sulfate and wereconcentrated under reduced pressure. The crude product was purified byflash column chromatography (Silica Gel, 100% DCM to 10% MeOH/DCM) toafford 296 mg (58%) of the mixture of the phosphoramidate diastereomers602b.

HPLC (Method A, 254 nm); 2b, 3.80 min; LCMS (m/e=532 (M⁺+1), 512 (M⁺−F);¹H NMR (400 MHz, CD₃OD) δ 1.18 (m, 6H), 1.28 (m, 3H), 3.27 (s, 1H), 3.85(m, 1H), 4.28 (m, 3H), 4.47 (dd, 1H), 4.93 (m, 1H), 5.60 (d, 0.3H), 5.65(d, 0.67H), 5.96 (m, 1H), 7.18 (m, 3H), 7.32 (m, 2H), 7.51 (d, 1H); ¹⁹FNMR (376 MHz, CD₃OD) δ −123.73 (m, 2.2F), −123.96 (m, 1F); ³¹P NMR (162MHz, CD₃OD), 3.43 (m, 2.2P), 3.59 (m, 1P).

Step 10: Semi-Preparative HPLC Separation of the Diastereomers of 602b

The mixture of diastereomers 602b was separated using a Phenomenex LunaC18 (2) and PrepMethod A. Approximately 290 mg of 602b was dissolved in2 mL of methanol/heptanes (80:20) to give a 145 mg/mL solution. Four 500μL injections were made. The fractions from the separations wereanalyzed by analytical HPLC (Method B). The suitable fractions werecombined and concentrated providing 50 mg (34%) of 602b diastereomer 1(13.99 min, 97.6 A %, >99.9% de) and 30 mg (20%) of 602b diastereomer 2(19.50 min, 96.8 A %, 94.2% de).

PrepMethod A:

Gilson prep HPLC system with GX-281 liquid handler and 322 pump.Phenomenex Luna C18(2) column, 150×21.20 mm, 5 μm. Mobile phase 40/60MeOH/water. Flow=22 ml/min.

HPLC Method B:

Luna C18 (2), 5 μm, 3.0×150 mm. Mobile Phase: 45% Methanol:Water(Isocratic). Flow=0.6 mL min⁻¹, 25 min runtime. DAD detector monitoredat 214 and 260 nm.

HPLC Method A:

Agilent Technologies 1100 Series HPLC with diode array detector. MobilePhase: ACN/NH₄OAc pH 4.4 buffer (5% to 80% over 10 min); Flow=1.4 mlmin⁻¹. DAD detector monitored at 254 and 272 nm.

Compound 603a

Step 1:

The nucleoside C1 (10 g, 28.3 mmol) was dissolved in a 1:1 mixture ofdimethoxypropane (50 mL, 408 mmol, 14.4 eq) and dimethylformamide (DMF,50 mL). p-Toluenesulfonic acid monohydrate (p-TSA, 2.05 g, 10.77 mmol,0.380 eq) was added and the mixture was stirred at room temperature for48 hours. Initially, 0.1 eq of p-TSA was added; after 24 hours, thereaction was only 50% complete. Additional aliquots of p-TSA (0.28 eqtotal) were needed to drive the reaction to completion. The reactionmixture was concentrated on a rotary evaporator and the residue wasdissolved in dichloromethane (DCM, 300 mL). The mixture was transferredto a separatory funnel and was washed with saturated sodium bicarbonatesolution (300 mL). The aqueous phase was back-extracted with 2×100 mL ofDCM and the combined organic phases were dried over magnesium sulfateand were concentrated under reduced pressure affording the crude productC2 (1.2 g, 108%). (¹H NMR analysis showed that the crude productcontained DMF).

HPLC (Method A, 254 nm), RT 3.4 min; LCMS (M⁻−1 m/e=392) ¹H NMR (400MHz, CDCl₃) δ 12.11 (br s, 1H), 7.95 (s, 1H), 7.82 (s, 1H), 5.80 (d,1H), 5.08 (dd, 1H), 4.94 (dd, 1H), 4.31 (m, 1H), 3.84 (m, 1H), 3.70 (m,1H), 2.65 (sept, 1H), 2.37 (br s, 1H), 1.51 (s, 3H), 1.28 (s, 3H), 1.18(a-t, 6H).

Step 2:

The crude nucleoside C2 (12.1 g, 28.3 mmol) was dissolved indichloromethane (DCM, 125 mL) under argon. Dimethylaminopyridine (DMAP,8.6 g, 70.8 mmol, 2.5 eq) was added and the mixture was cooled in anice-bath. Tosyl chloride (TsCl, 7.0 g, 36.8 mmol, 1.3 eq) was added andthe mixture was stirred at 0° C. for 30 minutes. The ice-bath wasremoved and the mixture was allowed to stir at room temperature for anadditional 30 minutes. HPLC analysis showed that the reaction wascomplete. The mixture was transferred to a separatory funnel and wasdiluted with DCM (125 mL). The DCM solution was washed with 1M HCl(2×100 mL), saturated bicarbonate solution (100 mL), and brine (100 mL).The mixture was dried over magnesium sulfate and was concentrated underreduced pressure affording 15.73 g of the desired product C3 (101%,contains DMF).

HPLC (Method A, 254 nm), RT 4.78 min; LCMS (M⁺+1, m/e=548); ¹H NMR (400MHz, CDCl₃) δ 12.11 (br s, 1H) 9.20 (br s, 1H), 7.66 (d, 2H), 7.58 (s,1H), 7.27 (d, 2H), 5.79 (d, 1H), 5.22 (dd, 1H), 5.12 (dd, 1H), 4.49 (dd,1H), 4.33 (m, 1H), 4.05 (dd, 1H), 2.61 (sept, 1H), 2.38 (s, 3H), 1.52(s, 3H), 1.31 (s, 3H), 1.18 (d, 3H), 1.14 (d, 3H).

Step 3:

The nucleoside C3 (8.0 g, 14.6 mmol) was dissolved in pyridine (80 mL)under an argon atmosphere. Diisopropylethylamine (DIPEA, 5.08 mL, 29.2mmol, 2 eq)) was added followed by diphenylcarbamoyl chloride (DPC-Cl,3.72 g, 1.1 eq). The mixture was stirred at room temperature under anargon atmosphere for 1 hour. HPLC analysis indicated the reaction to becomplete. The mixture was quenched by the addition of water (15 mL) andwas concentrated under reduced pressure. The residue was transferred toa separatory funnel with DCM (150 mL). The DCM solution was washed withaqueous HCl (1M, 100 mL), dried over magnesium sulfate, and wasconcentrated under reduced pressure. The crude product was purified byflash column chromatography (silica gel, 0→50% EtOAc/heptanes) toprovide 9.5 g of C4 (87%).

HPLC (Method A, 254 nm), RT 6.53 min; LCMS (M⁺+1, m/e=743); ¹H NMR (400MHz, CDCl₃) δ 7.99 (br s, 1H), 7.81 (s, 1H), 7.35 (m, 12H), 6.92 (d,2H), 5.91 (d, 1H), 5.42 (dd, 1H), 5.14 (dd, 1H), 4.40 (m, 1H), 4.29 (m,2H), 2.61 (sept, 1H), 2.10 (s, 3H), 1.50 (s, 3H), 1.29 (s, 3H), 1.18(2d, 6H).

Step 4:

The nucleoside C4 (9.5 g, 12.8 mmol) was dissolved in acetone (100 mL)under an argon atmosphere. Sodium iodide (13.4 g, 89.6 mmol, 7 eq) wasadded and the mixture was refluxed overnight. LCMS analysis indicatedthat the reaction was complete. The mixture was allowed to cool and wasconcentrated under reduced pressure. The mixture was transferred to areparatory funnel with DCM (100 mL) and was washed with a mixture of 5%sodium bicarbonate and 5% sodium thiosulfate (75 mL total). The organicphase was dried over magnesium sulfate and concentrated under reducedpressure affording 9 grams of a dark-colored foam. The crude materialwas purified by flash column chromatography (silica gel, 0→50%EtOAc/heptanes) to provide 7.82 g of C5 (88%).

HPLC (Method A, 254 nm), RT 6.39 min; LCMS (M⁺+1, m/e=699); ¹H NMR (400MHz, CDCl₃) δ 7.96 (s, 1H), 7.95 (br s, 1H), 7.30 (m, 10H), 6.00 (d,1H), 5.40 (m, 2H), 4.40 (m, 1H), 3.45 (m, 1H), 3.20 (dd, 1H), 2.67 (m,1H), 1.54 (s, 3H), 1.34 (s, 3H), 1.19 (m, 6H).

Step 5:

The nucleoside C5 (7.82 g, 11.2 mmol) was dissolved in toluene.1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 5.0 mL, 33.6 mmol, 3 eq) wasadded dropwise over 3 minutes. The mixture was stirred at roomtemperature for ca 64 hours. HPLC analysis indicated the reaction to becomplete. The reaction mixture was diluted with DCM (50 mL) andsaturated sodium bicarbonate solution (50 mL). This mixture wastransferred to a reparatory funnel along with additional portions of DCM(100 mL) and saturated sodium bicarbonate solution (50 mL). The layerswere separated and the organic phase dried over magnesium sulfate andwas concentrated under reduced pressure. The crude product was purifiedby flash column chromatography (silica gel, 0→4% MeOH/DCM) affording3.83 g of the desired product C6 (60%).

HPLC (Method A, 254 nm), RT 6.04 min; LCMS (M⁺+1, m/e=571); ¹H NMR (400MHz, CDCl₃) δ 7.88 (s, 1H), 7.87 (br s, 1H), 7.27 (m, 10H), 6.11 (s,1H), 5.88 (d, 1H), 5.27 (d, 1H), 4.48 (m, 1H), 4.39 (m, 1H), 2.75 (m,1H), 1.50 (s, 3H), 1.38 (s, 3H), 1.19 (m, 6H).

Step 6:

The nucleoside C6 (1.1 g, 1.9 mmol) was dissolved in DCM (10 mL).Freshly crushed silver fluoride (1.22 g, 9.6 mmol, 5 eq) was added. In aseparate flask, iodine (627 mg, 2.5 mmol, 1.3 eq) was dissolved in DCM(10 mL). The iodine solution was added drop-wise to the solution of thenucleoside over 30 minutes. After stirring for 5 minutes, HPLC analysisindicated that the reaction was incomplete. An additional 5 eq ofcrushed silver fluoride (1.22 g, 9.6 mmol) was added followed by theportion-wise addition of solid iodide (0.5 eq, 125 mg) over 5 minutes.After stirring at room temperature for 5 minutes, HPLC analysis showedthat the reaction was complete. The mixture was quenched by the additionof 20 mL of a mixture of 5% sodium bicarbonate and 5% sodiumthiosulfate. The mixture was filtered through Celite™ and wastransferred to a reparatory funnel. (Some finely divided solids were notremoved by the Celite™ filtration and were present in the organicphase.) The organic solution was dried over magnesium sulfate and wasconcentrated under reduced pressure providing the crude product (1.45g). The crude product was a 2:1 mixture of C7a and C7b. The crudeproduct was purified by flash column chromatography (silica gel, 0→50%EtOAc) affording 396 mg of the desired diastereomer C7a (29%). Inanother reaction, the desired diastereomer, C7a, was obtained in 57%yield after chromatography accompanied by a 21% isolated yield of the “Sdiastereomer”.

HPLC (Method A, 254 nm), RT 6.47 min; LCMS (M⁺+1, m/e=717); ¹H NMR (400MHz, CDCl₃) 8.01 (br s, 1H), 7.89 (s, 1H), 7.34 (m, 10H), 6.27 (s, 1H),6.10 (dd, 1H), 5.10 (d, 1H), 3.70 (m, 1H), 3.66 (s, 1H), 2.61 (sept,1H), 1.58 (s, 3H), 1.32 (s, 3H), 1.20 (m, 6H); ¹⁹F NMR (376 MHz, CDCl₃);δ −101.14 (m, 1F).

Step 7:

The nucleoside C5 (837 mg, 1.17 mmol) was dissolved in DCM (17 mL). In aseparate flask, a solution of potassium hydrogen phosphate (306 mg, 1.76mmol, 1.5 eq) in water (1 mL) was prepared. This solution along withbis(tetrabutylammonium) sulfate (50% in water, 2.34 mL, 1.17 mmol, 131)were added to the solution of the nucleoside. m-Chloroperbenzoic acid(mCPBA, 1.21 g, 7.02 mmol, 6 eq) was added and the mixture was stirredrapidly at room temperature overnight. HPLC analysis indicated thereaction to be complete. The mixture was transferred to a reparatoryfunnel and was washed with a mixture of 5% sodium bicarbonate and 5%sodium thiosulfate (20 mL total volume). The layers were separated andthe organic phase was dried over magnesium sulfate and was concentratedunder reduced pressure. The crude product was purified by flash columnchromatography (silica gel, 0→50% EtOAc/heptane) providing the desiredproduct C8 (525 mg, 60%).

HPLC (Method A, 254 nm), RT 6.93 min; LCMS (M⁺+1, m/e=745); ¹H NMR (400MHz, CDCl₃) δ 8.15 (br s, 1H), 7.93 (m, 1H), 7.91 (s, 1H), 7.82 (m, 1H),7.20-7.55 (m, 12H), 6.25 (s, 1H), 6.11 (dd, 1H), 5.11 (d, 1H), 4.66 (dd,1H), 4.49 (a-t, 1H), 2.49 (m, 1H), 1.59 (s, 3H), 1.35 (s, 3H), 1.07 (m,6H); ¹⁹F NMR (376 MHz, CDCl₃); δ −110.89 (m, 1F).

Step 8:

The nucleoside C8 (1.92 g, 2.58 mmol) was dissolved in n-butyl amine (19mL) forming a green-colored solution. The mixture was stirred and heatedto 80° C. for 30 minutes. (The color of the solution had turned red).HPLC analysis indicated the reaction to be complete. The mixture wasconcentrated under reduced pressure. DCM (20 mL) was added to the redoil forming a thick precipitate. The precipitate was removed byfiltration and was washed with copious amounts of cold DCM providing awhite-colored solid. This solid was dried in a vacuum oven overnightaffording 538 mg of the desired product C9 (61%)

HPLC (Method A, 254 nm), RT 2.66 min; LCMS (M⁻−1, m/e=340); ¹H NMR (400MHz, MeOD) δ 7.87 (s, 1H), 6.32 (s, 1H), 5.44 (dd, 1H), 5.17 (dd, 1H),3.75 (s, 1H), 3.73 (s, 1H), 1.58 (s, 3H), 1.38 (s, 3H); ¹⁹F NMR (376MHz, CDCl₃); δ −116.90 (m, 1F).

Step 9:

Preparation of (2R)-isopropyl2-((chloro(phenoxy)phosphoryl)amino)propanoate, C12. Phenyldichlorophosphate (437 μL, 2.93 mmol) was dissolved in THF (4 mL) andwas cooled to −66° C. with dry-ice/acetone. In a separate flask, D-Alaisopropyl ester (516 mg, 3.08 mmol, 1.05 eq) was dissolved in DCM (5mL). This solution was added to the solution of the dichlorophosphatedrop wise over 5 minutes. Triethylamine (855 μL, 6.15 mmol, 2.1 eq) wasadded drop wise over 5 minutes and the mixture was stirred for 30minutes at −66° C. The formation of the chlorophosphoramidate reagentC12 was shown to be complete by ¹H NMR, ³¹P NMR and LCMS.

LCMS (M⁻−Cl+OH−1, m/e=286); ³¹P NMR (162 MHz, CDCl₃), δ 8.08 (1P), 7.72(1P).

The nucleoside C9 (500 mg, 1.46 mmol, 0.5 eq) was suspended in THF (5mL) and was cooled to −66° C. t-Butyl magnesium chloride (1 M in THF,3.22 mL, 3.22 mmol, 1.1 eq) was slowly added over 5 minutes. The mixturewas stirred for 5 minutes followed by the addition of thechlorophosphate C12 (prepared above) over 8 minutes. The dry-ice bathwas replaced with an ice-bath and the reaction mixture was stirred at 0°C. for 30 minutes. HPLC analysis indicated the reaction to be complete.The mixture was quenched by the addition of 20% sodium chloride (NaCl,25 mL) and was extracted with DCM (2×10 mL). The organic solution waswashed with brine (25 mL), dried over MgSO4 and was concentrated. Thecrude product was purified by flash column chromatography (0→10%MeOH/DCM) to provide 317 mg of C10 (36%) as a single diastereomer. Latercolumn cuts provided an additional 365 mg (41%) of product which was 85%pure.

HPLC (Method A, 254 nm), RT 6.26 min; LCMS (M⁺+1, m/e=611).

Step 10:

The nucleoside C10 (315 mg, 0.52 mmol) was dissolved in 80% formic acid(15 mL) and was allowed to stir at room temperature for 15 hours. HPLCanalysis showed the reaction to be complete. The mixture wasconcentrated under reduced pressure and the crude material was purifiedby flash column-chromatography (silica gel, 0→10% MeOH/DCM) to afford168 mg of 3a (57%) as a single diastereomer.

HPLC (Method A, 254 nm), RT 3.52 min; LCMS (M⁺+1, m/e=571); ¹H NMR (400MHz, DMSO-d₆) δ 10.70 (br s, 1H), 7.84 (s, 1H), 7.33 (m, 2H), 7.16 (m,3H), 6.56 (br s, 2H), 6.03 (m, 2H), 5.92 (br s, 1H), 5.35 (br s, 1H),4.83 (m, 1H), 4.65 (dd, 1H), 4.44 (m, 1H), 4.19 (m, 2H), 3.71 (m, 1H),1.21 (m, 3H), 1.14 (m, 6H); ¹⁹F NMR (DMSO-d₆, 376 MHz,); δ −120.7 (m,1F); ³¹P NMR (162 MHz, DMSO-d₆), δ 3.53 (1P).

HPLC Method A:

Agilent Technologies 1100 Series HPLC with diode array detector. MobilePhase: ACN/NH₄OAc pH 4.4 buffer (5% to 80% over 10 min). Flow=1.4 mlmin⁻¹. DAD detector monitored at 254 and 272 nm.

Example 1E Preparation of Diastereomerically Pure D-Alanine,N—((R_(P),2′R)-2′-deoxy-2′-fluoro-2′-methyl-P-phenyl-5′-uridylyl)-,1-methylethyl ester Compound (804ai)

To a stirred solution of D-Alanine isopropyl ester hydrochloride (47.7mmol) in anhydrous CH₂Cl₂ (1.05 mL/mmol) was added TEA (98.30 mmol) at−70° C. over 15 minutes dropwise. To this mixture was added a solutionof phenyl dichlorophosphate (47.7 mmol) in anhydrous CH₂Cl₂ (1.05mL/mmol) over 1 hour. The reaction mixture was stirred at thistemperature for additional 30 minutes and then allowed to warm to 0° C.over 2 hours. To this mixture was added a solution of pentafluorophenol(47.7 mmol) and TEA (52 mmol) in CH₂Cl₂ (50 mL). The reaction mixturewas stirred at 0° C. during 1 hour. The triethylamine salt was filteredwashed with CH₂Cl₂. The filtrate was concentrated under reducedpressure, the residue was triturated with TBME (150 mL). Theheterogeneous mixture was filtered and the solid was rinsed with TBME.The filtrate was concentrated and the residue was triturated with amixture of hexane/ethyl acetate 20% (100 mL). The suspension wasfiltered and the solid was rinsed with a mixture of hexane/ethyl acetate20% and dried to give the expected compound 1 in 11% yield as a singleisomer.

¹H NMR (400 MHz, CDCl₃): δ (ppm) 1.23-1.26 (m, 6H), 1.46 (d, J=7.02 Hz,3H), 3.94 (dd, J=9.47 Hz and 12. Hz, 1H), 4.09-4.19 (m, 1H), 4.99-5.09(m, 1H), 7.19-7.27 (m, 3H), 7.34-7.38 (m, 2H).

D-Alanine,N—((R_(P),2′R)-2′-deoxy-2′-fluoro-2′-methyl-P-phenyl-5′-uridylyl)-,1-methylethyl ester (804ai)

Compound 2 was prepared according to published procedures. To a solutionof compound 2 (4.23 mmol) in THF (3.92 mL/mmol) at −5° C. under nitrogenwas added dropwise tert-butylmagnesium chloride (1M in THF) (8.92 mmol).The heterogeneous reaction mixture was stirred during 30 minutes at −5°C. and 30 minutes at room temperature. The reaction mixture was cooleddown to −5° C. under nitrogen and compound 1 (5.07 mmol) in THF (18 mL)was added dropwise. The reaction mixture was stirred at −5° C. to 0° C.overnight. The reaction mixture was quenched with aqueous solution ofHCl 1N (20 mL) at −5° C. and extracted with CH₂Cl₂. The organic layerwas washed with H₂O, Na₂CO₃ aq 5%, H₂O and brine. The organic layer wasdried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by chromatography on silica gel (eluent: 100%CH₂Cl₂ to CH₂Cl₂:CH₃OH 95:5) to give the desired pure isomer as a whitepowder in 77% yield.

The crystal structure of pure isomer was obtained. The crystal structureshowed the pure isomer corresponds to the R_(P) isomer of Formula 804ai.

¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.25 (d, J=6.26 Hz, 6H), 1.33 (d,J=22.32 Hz, 3H), 1.38 (d, J=6.97 Hz, 3H), 3.61-3.63 (m, 1H), 3.72-3.98(m, 3H), 4.06-4.10 (m, 1H), 4.39-4.51 (m, 2H), 5.03 (sept, J=6.22 Hz,1H), 5.58 (dd, J=2.29 Hz and 8.19 Hz, 1H), 6.16 (d, J=19.05 Hz, 1H),7.19-7.26 (m, 4H), 7.34-7.38 (m, 2H), 8.43 (brs, 1H); ³¹P NMR (161.98MHz, CDCl₃) δ (ppm) 4.29 (s, 1P). LCMS (ESI+) m/z 530.2 [M+H]⁺ 100%.LCMS (ESI−) m/z 528.2 [M−H]⁻ 100%.

Example 1F Preparation of 2′-cyano, azido and amino nucleosides

Ethyl 2-cyano-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylbutanoate (A2)

A 5 L flange flask was fitted with a thermometer, nitrogen inlet,pressure equalizing dropping funnel, bubbler, and a subaseal. Methyllithium solution (956 mL, 1.6M in diethylether, 1.7 equiv.) was added,and the solution was cooled to about −25° C. Diisopropyl amine (214 mL,1.7 equiv.) was added using the dropping funnel over about 40 minutes.The reaction was left stirring, allowing to warm to ambient temperatureovernight. CO_(2(s))/acetone cooling was applied to the LDA solution,cooling to about −70° C. R-Glyceraldehyde dimethylacetal solution (50%in DCM) was evaporated down to ˜100 mbar at a bath temp of 35° C., toremove the DCM, then azeotroped with anhydrous hexane (2×100 mL), undervacuum. The fresh aldehyde (120 g, 0.9 mol) and ethyl2-cyanopropionionate (170 mL, 1.5 equiv.) were placed in a 1 L roundbottom flask, which was filled with toluene (800 mL). This solution wascooled in a CO_(2(s))/acetone bath, and added via cannula to the LDAsolution over about 50 minutes, keeping the internal temperature of thereaction mixture cooler than −55° C. The mixture was stirred withcooling (internal temp. slowly fell to ˜−72° C.) for 90 min, then warmedto room temperature over 30 minutes using a water bath. This solutionwas added to a sodium dihydrogen phosphate solution 300 g of NaH₂PO₄ in1.5 L of ice/water, over about 10 minutes, with ice-bath cooling. Themixture was stirred for 20 minutes, then filtered and transferred to areparatory funnel, and partitioned. The solid was further washed withEtOAc (2×1 L) and the washings were used to extract the aqueous. Thecombined organic extracts were dried over sodium sulfate. The volatileswere removed in vacuo. The resultant oil was hydrolyzed crude.

3-Cyano-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one

The crude oil was taken up in acetic acid (1.5 L, 66% in water) andheated to 90° C. over one hour, then at held at that temperature for onehour. Once the mixture had cooled to room temperature, the volatileswere removed in vacuo, and azeotroped with toluene (2×500 mL). Theresultant oil was combined with some mixed material from an earliersynthesis and columned in two portions (each ˜1.25 L of silica,0→12.5%→25→50% EtOAc in DCM). The lower of the two main spots is thedesired material; fractions containing this material as the majorcomponent were combined and the solvent removed in vacuo to give 85.4 gof a brown oil as a mixture of 3 diastereomers (15:8:2).

((2R,3S,4R)-3-(benzoyloxy)-4-cyano-4-methyl-5-oxotetrahydrofuran-2-yl)methylbenzoate (A5)

A 2 L 3-neck round bottom flask was fitted with an overhead stirrer,thermometer and pressure equalizing dropping funnel under nitrogen.3-Cyano-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one (85.4 g, 0.50mol) in acetonitrile (1.5 L) was added, followed by4-dimethylaminopyridine (700 mg) and benzoyl chloride (128 mL, 2.2equiv.). Finally triethylamine (167 mL, 2.4 equiv.) was added over 10minutes using the dropping funnel. The addition of the triethylamine isaccompanied by a mild exotherm, which obviated the addition of a coldwater bath to keep the internal temperature below 25° C. The reactionwas stirred at ambient temperature for 2.5 hours. The reaction mixturewas transferred to a separating funnel with EtOAc (2.5 L) and halfsaturated brine (2.5 L), and partitioned. The aqueous layer wasre-extracted with EtOAc (1.5 L). The combined organic layers were washedwith 50% Sodium bicarbonate/25% Brine (1.5 L) and dried over sodiumsulphate. The resultant brown solid was twice recrystallized fromhexane/chloroform, to give ˜15 g of product of the desired purity. Themother liquors from the recrystallizations were further recrystallizedfrom chloroform/hexanes several times to give a further 15 g of product.

¹H NMR (CDCl₃, 400 MHz) δ (ppm) 8.11 (dm, J=8.3 Hz, 2H), 7.98 (dm, J=8.4Hz, 2H), 7.66 (tm, J=7.5 Hz, 1H), 7.59 (tm, J=7.5 Hz, 1H), 7.49 (tm,J=7.6 Hz, 2H), 7.43 (tm, J=7.6 Hz, 2H), 5.54 (d, J=6.5 Hz, 1H),4.97-5.02 (m, 1H), 4.77 (dd, J=12.7, 3.5 Hz, 1H), 4.66 (dd, J=12.7, 4.7Hz, 1H), 1.88 (s, 3H).

3,5-Di-O-benzoyl-2-C-cyano-2-C-methyl-D-ribofuranose (A6)

To a solution of A5 (81.08 mmol) in anhydrous tetrahydrofuran (650 mL)was added under inert atmosphere at −35° C., LiAlH(OtBu)₃ (1.0 M intetrahydrofuran, 21.7 mmol) over a 20 minutes period. The reactionmixture was stirred for 1 hour at −20° C. and quenched by addition of asaturated NH₄Cl solution, keeping the temperature bellow 0° C. Ethylacetate was added and the white suspension was filtered through a pad ofcelite and washed with ethyl acetate. The filtrate was extracted withethyl acetate twice. The combined organic layers were dried overanhydrous sodium sulfate, filtered and evaporated under reducedpressure. The expected intermediate was used without furtherpurification for the next step. MS (ESI) m/z=404 (MNa⁺).

1-O-Acetyl-3,5-di-O-benzoyl-2-C-cyano-2-C-methyl-D-arabinofuranose (A7)

To a solution of A6 (81.0 mmol) in anhydrous tetrahydrofuran (420 mL)was added dropwise under inert atmosphere (nitrogen) at 0° C., aceticanhydride (405.0 mmol) followed by 4-dimethylaminopyridine (8.1 mmol).The reaction mixture was allowed to warm-up to room temperature and wasstirred for 1 hour. The crude was partially concentrated under reducedpressure, partitioned with dichloromethane and a saturated NaHCO₃solution, then transferred into a separatory funnel. The organic layerwas extracted, dried, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography on silica gel [eluent:petroleum ether/ethyl acetate: 0 to 100%] to afford a α,β sugar mixtureA7 in 96% overall yield (2 steps). MS (ESI) m/z=869.2 (2MNa⁺).

3′,5′-Di-O-benzoyl-2′-C-cyano-2′-C-methyl-4-benzoyl-α,β-cytidine (A8)

To a suspension of N-benzoyl cytosine (23.62 mmol), and a catalyticamount of ammonium sulfate in 4-chlorobenzene (60 mL) was added HMDS(70.85 mmol). The reaction mixture was heated at 140° C. overnight. Thesolvent was removed under inert atmosphere and the residue was taken in4-chlorobenzene (20 ml). Then, 7 (11.81 mmol) in chlorobenzene (40 mL)was added dropwise to the reactional mixture followed by SnCl₄ (23.62mmol) dropwise. The reaction mixture was stirred at 70° C. overnight,cooled to room temperature and diluted with dichloromethane and asaturated NaHCO₃ solution. The white suspension was filtered through apad of celite and washed with dichloromethane. The filtrate wasextracted with dichloromethane twice. The combined organic layers weredried over anhydrous Na₂SO₄, filtered and evaporated under reducedpressure to afford expected nucleoside as an α,β mixture. Crude materialwas used without further purification for the next step. MS (ESI)m/z=598.2 (MH⁺).

2′-C-Cyano-2′-C-methyl-α,β-cytidine, hydrochloride form (A9)

A suspension of A8 (11.8 mmol) in 7N methanolic ammonia (150 mL) wasstirred at room temperature for 3 days in a stainless steel pressurereactor. The mixture was evaporated to dryness, diluted with water andtransferred into a reparatory funnel. The aqueous layer was extractedwith dichloromethane and water was removed under reduced pressure. Cruderesidue was diluted with ethanol (50 mL) and 10 mL of 1.25 N HCl indioxan were added. Concentration of the reaction mixture under reducedpressure followed by 3 co-evaporations with absolute ethanol afforded aprecipitate which was filtrated-off and washed with absolute ethanol togive pure expected compound as a white solid in 41% overall yield (2steps) (57/43 α,β mixture).

¹H NMR (DMSO, 400 MHz) δ (ppm) 1.15 (s, 3Hβ), 1.51 (s, 3Hα), 3.45-3.95(m, 3Hα,β), 4.00-4.10 (m, 1Hα,β), 4.98 (brs, 1Hα), 5.29 (brs, 1Hβ), 5.80(d, J=7.40 Hz, 1Hβ), 5.89 (d, J=7.40 Hz, 1Hα), 5.95 (s, 1Hα), 6.22 (s,1Hβ), 6.42 (brd, 1Hα,β), 7.53 (brs, 1Hα,β), 7.76 (d, J=7.40 Hz, 1Hα),7.89 (brs, 1Hα,β), 7.96 (d, J=7.40 Hz, 1Hβ); MS (ESI) m/z=267 (MH⁺).

Compound A9b: The white solid A9 was triturated with a mixture ofmethanol/triethylamine/water; and filtered to afford an off-white solidA9a as α-anomer, and a filtrate. The filtrate was concentrated underreduced pressure and purified by flash chromatography on silica gel[eluent: DCM/methanol: 80/20, with 1% of Et3N] to afford the expectedβ-anomer A9b. Off-white solid, ¹H NMR (DMSO, 400 MHz) δ (ppm) 1.13 (s,3H), 3.60-3.65 (m, 1H), 3.77-3.90 (m, 3H), 5.26 (brt, 1H), 5.73 (d,J=7.42 Hz, 1H), 6.24 (s, 1H), 6.38 (brd, 1H), 7.29 (brd, 2H), 7.88 (d,J=7.42 Hz, 1H); MS (ESI) m/z=267 (MH⁺).

Compound A10

To a solution of compound A9 (2.31 mmol) in dry pyridine (16 mL) and DMF(was added dropwise TIPSCl₂ (2.54 mmol) under nitrogen atmosphere. Thereaction was stirred for 5 hours at room temperature. Then, DMAP (2.31mmol) and mMTrCl (2.77 mmol) were added at room temperature and thereaction mixture was stirred at 55° C. overnight. The reaction mixturewas slowly added to a saturated solution of NaHCO₃. The aqueous layerwas extracted with DCM and the combined organic layers were dried overNa₂SO₄, filtered and concentrated under reduced pressure. The crude wasdiluted in MeOH (16 mL) and NH₄F (11.55 mmol) was added. The reactionmixture was stirred at 60° C. during 1.5 hour and concentrated underreduced pressure. The residue was purified by flash chromatography onsilica gel [eluent: DCM to DCM/MeOH 95/5] to afford a mixture ofexpected β nucleoside 10 (270 mg, beige foam, 22% overall yield) and αnucleoside A11 (416 mg).

Compound A10: δ (ppm) 0.97 (s, 3H), 3.51-3.87 (m, 4H), 3.71 (s, 3H),5.23 (brt, 1H), 6.06 (s, 1H), 6.26 (d, J=7.50 Hz, 1H), 6.35 (brd, J=4.50Hz, 1H), 5.80 (d, J=7.40 Hz, 1Hβ), 6.80-7.40 (m, 14H), 7.80 (d, J=7.50Hz, 1H), 8.51 (brs, 1H); MS (ESI) m/z=537.2 (MH⁻).

Compound A12

To as solution of compound A10 (0.39 mmol) in anhydrous THF (2 mL) undernitrogen at −5° C. was added dropwise tert-butylmagnesium chloride (1.0Min THF) (0.82 mmol). The white suspension was stirred at thistemperature for 15 minutes and then warmed to ambient temperature andstirred for an additional 20 minutes. The reaction mixture was cooleddown to 0° C. and compound A12.0 (0.47 mmol) solubilized in THF (2 mL)was added dropwise. DMSO (0.4 mL) was added and the mixture was stirredat 7° C. overnight. The reaction mixture was diluted withdichloromethane and washed with H₂O. The organic phase was dried,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on silica gel [eluent: DCM/MeOH 0 to4%] to give the expected compound in 68% yield. MS (ESI) m/z=806.2(MH⁻).

Compound 901

To a solution of compound A12 (0.27 mmol) in DCM (10 mL) was addeddropwise TFA (2.67 mmol) under nitrogen. The reaction mixture wasstirred at room temperature overnight. The reaction mixture was purifieddirectly by flash chromatography on silica gel and by preparative HPLCto give the expected compound as a white powder.

¹H NMR (DMSO, 400 MHz) δ (ppm) 1.15 (d, J=3.06 Hz, 3H), 1.17 (d, J=3.06Hz, 3H), 1.25 (brd, 6H), 3.65 (brd, J=13.0 Hz, 1H), 3.77-3.91 (m, 2H),4.00 (brd, J=7.22 Hz, 1H), 4.70 (t, J=8.30 Hz, 1H), 4.88 (heptuplet,J=6.30 Hz, 1H), 5.28 (brs, 1H), 5.76 (d, J=7.52 Hz, 1H), 6.28 (s, 1H),6.34 (q, J=10.30 Hz, J=10.36 Hz), 7.17-7.27 (m, 3H), 7.31-7.42 (m, 4H),7.83 (d, J=7.57 Hz, 1H); ³¹P NMR (DMSO, 161.98 MHz): δ (ppm) 3.48 (s,1P); MS (ESI) m/z=536.2 (MH⁺).

Example 2 HCV Replicon Assay

Huh-7-derived cell line (Zluc) that harbors an HCV genotype 1b repliconand a luciferase reporter gene was grown in Dulbecco's Modified EagleMedium (DMEM) supplemented with 10% fetal bovine serum, 2 mM GlutaMAX,1% MEM nonessential amino acids, 100 IU/mL penicillin, 100 μg/mLstreptomycin, and 0.5 mg/mL Geneticin® (G418). For dose response testingthe cells were seeded in 96-well plates at 7.5×10³ cells per well in avolume of 50 μL, and incubated at 37° C./5% CO₂. Drug solutions weremade up freshly in Huh-7 media as 2× stocks. Ten additional 5-folddilutions were prepared from these stocks in DMEM without G418. At leastthree hours after Zluc cells were seeded, drug treatment was initiatedby adding 50 μL of drug dilutions to the plates in duplicate. Finalconcentrations of drug ranged from 100 μM to 0.0000512 μM. Cells werethen incubated at 37° C./5% CO₂. Alternatively, compounds were tested attwo concentrations (1 μM and 10 μM). In all cases, Huh-7 (which do notharbors the HCV replicon) served as negative control. After 72 hours ofincubation, the inhibition of HCV replication was measured byquantification of photons emitted after mono-oxygenation of5′-fluoroluciferin to oxyfluoroluciferin by firefly luciferase. Forthis, media was removed from the plates via gentle tapping. Fiftymicroliters of ONE-glo luciferase assay reagent was added to each well.The plates were shaken gently for 3 min at room temperature andluminescence was measured on a Victor³ V 1420 multilabel counter (PerkinElmer) with a 1 second read time using a 700 nm cut-off filter. The EC₅₀values were calculated from dose response curves from the resultingbest-fit equations determined by Microsoft Excel and XLfit 4.1 software.When screening at two fixed concentrations, the results were expressedas % inhibition at 1 μM and 10 μM.

For cytotoxicity evaluation, Zluc cells were treated with compound asdescribed herein, and cell viability was monitored using theCellTiter-Blue Cell Viability Assay (Promega) by adding 20 μL of theassay solution to each well. The plates were then incubated at 37° C./5%CO₂ for at least 3 hours. Fluorescence was detected in plates usingexcitation and emission wavelengths of 560 and 590 nm, respectively, ina Victor³ V 1420 multilabel counter (Perkin Elmer) and CC₅₀ values weredetermined using Microsoft Excel and XLfit 4.1 software.

Compounds presented in Table 2 below were assayed according to thereplicon assay described herein.

TABLE 2 HCV Replicon Activity HCV HCV Replicon Compound RepliconCompound Reference EC₅₀ CC₅₀ Reference EC₅₀ CC₅₀ Compound 40ii ++++ +Compound 40ii +++ + Diastereomer 1 Diastereomer 2 Compound 40i + +Compound 202i +++ + Diastereomer 1 Diastereomer 2 Compound 202i ++ +Compound 205i +++ + Diastereomer 1 Diastereomer 2 Compound 205i ++ +Compound 603a +++ ++ Diastereomer 1 Single Diastereomer Compound 401 ++++ Compound 401 +++ ++ Diastereomer 1 Diastereomer 2 Compound 425 + ++Compound 425 + ++ Diastereomer 1 Diastereomer 2 Compound 502a ++ ++Compound 502a + ++ Diastereomer 1 Diastereomer 2 Compound 602b + ++Compound 602b ++ ++ Diastereomer 1 Diastereomer 2 EC₅₀ is provided asfollows: ++++ ≦ 250 nM, 250 nM < +++ ≦ 1 μM, 1 μM < ++ ≦ 10 μM, and + >10 μM CC₅₀ is provided as follows: ++ ≦ 50 μM, + > 50 μM

Example 3 Metabolism Assays

Assay for the Release of Active Metabolite in Huh-7 Cells.

Huh-7 cells were plated in 1 mL culture medium (DMEM, containingglucose, L-glutamine and sodium pyruvate, 10% FBS, 100 IU/mL penicillin,100 μg/mL streptomycin, 2 mM GlutaMAX, 1% MEM nonessential amino acids)at the concentration 0.8, 0.4 and 0.2 million cells per well on 6 wellplates for 24, 48 and 72 hr treatment, respectively. Plated cells wereincubated overnight at 37° C. in an incubator.

The following morning test compound was diluted to 20 μM from a stocksolution in DMSO in fresh culture medium pre-warmed to 37° C. and 1 mLof the solution/well was added to cells. A final medium volume per wellwas 2.0 mL, test compound concentration in well was 10 μM and final DMSOconcentration was 0.1%.

After 24, 48 or 72 hr, the medium was carefully removed and cellmonolayers were washed twice with 2 mL ice-cold PBS per well. Followingthe last wash, all PBS was carefully removed and 1.0 mL of extractionsolution (ice-cold 70% methanol) added. The plate was tightly coveredwith Parafilm, plastic plate cover and Parafilm again and anintracellular content was extracted at −20° C. for 24 hr.

After 24 hr the extracts were transferred into polypropylene microfugetubes and dry on a refrigerated centrivap concentrator. Dry residueswere reconstituted in 250 μL of HPLC-grade water and centrifuged at16,000×g for 10 min. Aliquots (100 μL each) of the supernatants weretransferred into a 96 well plate and internal standard (4 ng/mL finalconcentration) was added as the internal standard (IS) for LC-MS/MSanalysis.

Abbreviations:

FHH=fresh human hepatocytes; Ms=Mouse; MsH=fresh mouse hepatocyte.

Assay for the Release of Active Metabolite in Primary Hepatocytes:

Plates of fresh human and mouse hepatocytes were obtained on ice. Themedium was removed and replaced with hepatocyte culture medium(William's E supplemented with penicillin-streptomycin, 1% L-glutamine,1% insulin-transferrin-selenium and 0.1 μM Dexamethasone (Invitrogen) orwith Invitro GRO HI medium complemented with Torpedo antibiotics(Celsius)). Cells were left overnight in an incubator at 37° C. toacclimatize to culture and the medium.

Hepatocyte incubations were conducted at a final volume of 0.5 mLhepatocyte culture medium/well (0.8 million cells/well for human and 0.5million cells/well for mouse; 12 well plate no overlay, collagen coat).Culture medium from overnight incubation of cells was removed andreplaced with fresh medium, pre-warmed to 37° C., containing 10 μM oftest compound from a stock solution in DMSO (final DMSO concentrationwas 0.1%). At each specific time point, incubation medium was removedand cell monolayers were carefully washed two times with ice-cold PBS.Following the last wash, all PBS was carefully removed and 1.0 mL ofextraction solution (ice-cold 70% methanol/30% water) added. Cells werescraped off and suspended in the extraction solution, transferred to 2mL polypropylene microfuge tubes and intracellular contents extractedovernight at −20° C.

After the overnight treatment the cellular extracts were prepared bycentrifugation at 16,000×g for 10 min to remove cellular debris. Theremaining sample was then dried using a refrigerated centrivapconcentrator. Dry extracts were reconstituted in 1000 μL of HPLC-gradewater and centrifuged at 16,000×g for 10 min. Aliquots (100 μL each) ofthe supernatant were transferred into a 96 well plate and internalstandard (4 ng/mL final concentration) was added as the internalstandard (IS) for LC-MS/MS analysis.

The incubation time points were 6, 24 and 48 hours for human hepatocytesand 1, 4, 8, 12 and 24 hours for mouse hepatocytes. Results are providedin Table 4 below.

TABLE 4 Formation of Active Metabolite in Huh-7 cells and HepatocytesCompound 40ii Compound 40ii Compound 40i Compound 40i Cells Diastereomer2 Diastereomer 1 Diastereomer 1 Diastereomer 2 Huh-7 TP C_(max) 294 123ND ND (pmol/mill cells) Huh-7 TP 240 25 ND ND (24 hr) Huh-7 TP 294 96 NDND (48 hr) Huh-7 TP 144 123 ND ND (72 hr) Huh-7 TP AUC 14544 4380 ND ND(pmol · hr/mill cells) FHH TP AUC 4934 ND ND ND (pmol · hr/mill cells)FHH TP C_(max) 197 ND ND ND (pmol/mill cells) FHH TP (6 hr) 197 ND ND NDFHH TP (24 hr) 89 ND ND ND FHH TP (48 hr) 59 ND ND ND MsH AUC 1794 ND4052 1073 (pmol · hr/mill cells) MsH C_(max) 87 198 89 (pmol/mill cells)^(a)ND = not determined ^(b)BLD = below limit of detection

Example 4 Pharmacokinetics of Plasma Nucleoside and Liver TriphosphateFollowing a Single Oral Dose in CD-1 Mice

Abbreviations:

Ms=Mouse; TP=triphosphate.

A single oral dose of Compound 1 at 10 mg/kg in PEG 200 (dose volume 5mL/kg) was administered to nine CD-1 male mice. Five untreated animalswere used for the collection of control plasma and liver. Terminalplasma and liver samples were collected from three animals per timepoint at 4, 12 and 24 hours post dose. Liver specimens were collectedfrom all animals immediately after the incision. Freezing forceps storedin liquid nitrogen were used to freeze the liver before excision.

Plasma samples were analyzed for nucleoside by LC-MS/MS. The internalstandard (IS) was either 2′-MeG-D3 or tiapride. For proteinprecipitation and extraction, each plasma sample (50 μL) was treatedwith 500 μL of 0.2% formic acid in acetonitrile and 20 μL of theinternal standard working solution. After vortexing and centrifugation,500 μL of the sample extracts were transferred to a new plate, driedunder N₂ at ˜28° C. and reconstituted with 75 μL of 0.2% FA in water.The extracts were chromatographed on an Aquasil C18 column using agradient system of 0.2% formic acid in water and acetonitrile. Theanalytes were detected and quantified by tandem mass spectrometry inpositive ion mode on an MDS Sciex API5000 equipped with a TurboIonspray® interface. The calibration range was 0.500 (LLOQ) to 200 ng/mLin mouse plasma.

Liver samples were analyzed for the active species nucleosidetriphosphate by LC-MS/MS. The triphosphate levels were assayed byhomogenizing (on ice) a known weight of mouse liver with 4× volume of0.95 M trichloroacetic acid (TCA). Internal standard solution was addedto the homogenate followed by neutralization with 20% ammonium hydroxidesolution and addition of 500 μL 1% formic acid. The tissue samples wereextracted by weak anion exchange solid phase extraction (SPE). Postextraction, the eluates were evaporated under nitrogen, followed byreconstitution before injection onto the LC-MS/MS system. The sampleswere chromatographed on a Luna NH₂ column using a gradient system ofammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water andacetonitrile (70:30). The analyte was detected and quantified by tandemmass spectrometry in positive ion mode on an API4000 equipped with aTurbo Ionspray® interface.

Results are provided in Table 5 below.

TABLE 4 Mouse plasma and liver pharmacokinetic parameters Ms Plasma MsPlasma nucleoside AUC Ms Liver TP nucleoside C_(max) (pmol · hr/mL at MsLiver TP AUC (pmol · hr/g Compound (pmol/mL at 1 μmol/kg) 1 μmol/kg)C_(max) (pmol/g at 1 μmol/kg) at 1 μmol/kg) Compound 40ii 86 970 71 840Diastereomer 1 Compound 40ii 99 1300 34 560 Diastereomer 2 Compound 40i94 1400 520 6200 Diastereomer 1 Compound 40i 64 1000 430 4400Diastereomer 2 Compound 202i — 1700 — 8400 Diastereomer 1 Compound 202i— 1700 — 7200 Diastereomer 2 Compound 202ii — — 160 1200 Diastereomer 1Compound 202ii — — 84 850 Diastereomer 2 Compound 205i — 1400 — 5600Diastereomer 1 Compound 205i — 1700 — 6900 Diastereomer 2 ND = Notdetermined; BLQ = below limit of quantitation.

Example 4A Pharmacokinetics of Plasma Nucleoside and Liver TriphosphateFollowing a Single Oral Dose in CD-1 Mice

Abbreviations:

Ms=Mouse; TP=triphosphate.

A single oral dose of test compound at 2 mg/kg and 10 mg/kg in PEG 200(dose volume 1 mL/kg and 5 mL/kg) was administered to nine CD-1 malemice. Five untreated animals were used for the collection of controlplasma and liver. Terminal plasma and liver samples were collected fromthree animals per time point at 4, 12 and 24 hours post dose. Liverspecimens were collected from all animals immediately after theincision. Freezing forceps stored in liquid nitrogen were used to freezethe liver before excision.

Plasma samples were analyzed for nucleoside by LC-MS/MS. The internalstandard (IS) was either 2′-MeG-D3 or tiapride. For proteinprecipitation and extraction, each plasma sample (50 μL) was treatedwith 500 μL of 0.2% formic acid in acetonitrile and 20 μL of theinternal standard working solution. After vortexing and centrifugation,500 μL of the sample extracts were transferred to a new plate, driedunder N₂ at ˜28° C. and reconstituted with 75 μL of 0.2% FA in water.The extracts were chromatographed on an Aquasil C18 column using agradient system of 0.2% formic acid in water and acetonitrile. Theanalytes were detected and quantified by tandem mass spectrometry inpositive ion mode on an MDS Sciex API5000 equipped with a TurboIonspray® interface. The calibration range was 0.500 (LLOQ) to 200 ng/mLin mouse plasma.

Liver samples were analyzed for the active species nucleosidetriphosphate by LC-MS/MS. The triphosphate levels were assayed byhomogenizing (on ice) a known weight of mouse liver with 4× volume of0.95 M trichloroacetic acid (TCA). Internal standard solution was addedto the homogenate followed by neutralization with 20% ammonium hydroxidesolution and addition of 500 μL 1% formic acid. The tissue samples wereextracted by weak anion exchange solid phase extraction (SPE). Postextraction, the eluates were evaporated under nitrogen, followed byreconstitution before injection onto the LC-MS/MS system. The sampleswere chromatographed on a Luna NH₂ column using a gradient system ofammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water andacetonitrile (70:30). The analyte was detected and quantified by tandemmass spectrometry in positive ion mode on an API4000 equipped with aTurbo Ionspray® interface.

Results are provided in Table 3 below.

TABLE 4A Mouse plasma and liver pharmacokinetic parameters Compound 425Compound 425 Cells Diastereomer 1 Diastereomer 2 Ms Plasma nucleosideAUC 46 26 (pmol · hr/mL after 2 mg/kg dose) Ms Liver TP AUC (pmol · hr/gat 3900 1900 after 2 mg/kg dose) Ms Plasma nucleoside AUC 130 75 (pmol ·hr/mL after 10 mg/kg dose) Ms Liver TP AUC (pmol · hr/g at 7500 2500after 10 mg/kg dose)

Example 4B Pharmacokinetics of Plasma Nucleoside and Liver TriphosphateFollowing a Single Oral Dose in CD-1 Mice

Abbreviations:

Ms=Mouse; TP=triphosphate;

A single oral dose of test compound at 10 mg/kg and/or 2 mg/kg in PEG200 (dose volume 5 mL/kg) was administered to nine CD-1 male mice. Fiveuntreated animals were used for the collection of control plasma andliver. Terminal plasma and liver samples were collected from threeanimals per time point at 4, 12 and 24 hours post dose. Liver specimenswere collected from all animals immediately after the incision. Freezingforceps stored in liquid nitrogen were used to freeze the liver beforeexcision. Only liver samples were analyzed for triphosphate levels.

Liver samples were analyzed for the active species nucleosidetriphosphate by LC-MS/MS. The triphosphate levels were assayed byhomogenizing (on ice) a known weight of mouse liver with 4× volume of0.95 M trichloroacetic acid (TCA) in water. Internal standard solutionwas added to the homogenate and mixed. Sample homogenates werecentrifuged at 16.1 krpm for 5 minutes. Supernatants were transferred to96 well plates and injected onto the LC-MS/MS system. The samples werechromatographed on a Luna NH₂ column using a gradient system of ammoniumacetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water and acetonitrile(70:30). The analyte was detected and quantified by tandem massspectrometry using the analyte specific MRM transition on an API4000equipped with a Turbo Ionspray® interface.

Results are provided in Table 3 below.

TABLE 4B Mouse liver pharmacokinetic parameters Compound 602b Compound602b Compound 603a Compound 603b Compound 603b Cells Diastereomer 1Diastereomer 2 Single Diastereomer Diastereomer 1 Diastereomer 2 MsLiver TP AUC 5500 3600 120 110 — (pmol · hr/g at 1 μmol/kg) following asingle 10 mg/kg dose Ms Liver TP AUC 4100 3200 — — — (pmol · hr/g at 1μmol/kg) following a single 2 mg/kg dose

Example 4C Plasma and Liver Pharmacokinetics Following a Single OralDose in CD-1 Mice

Abbreviations:

Ms=Mouse; 2′-Me-2′-F-U=2′-methyl-2′-fluorouridine; 2′-Me-2′-F-UTP=2′-methyl-2′-fluorouridine triphosphate;2′-F-2′-Me-G=2′-fluoro-2′-methyl-guanosine.

A single oral dose of test compound at 10 mg/kg for 804a or 25 mg/kg for804b in PEG 200 (dose volume 5 mL/kg) was administered to nine CD-1 malemice. Five untreated animals were used for the collection of controlplasma and liver. Terminal plasma and liver samples were collected fromthree animals per time point at 4, 12 and 24 hours post dose. Liverspecimens were collected from all animals immediately after theincision. Freezing forceps stored in liquid nitrogen were used to freezethe liver before excision.

Plasma samples were analyzed for 2′-methyl-2′-fluorouridine(2′-Me-2′-F-U) by LC-MS/MS. The internal standard (IS) wasD₃-2′-F-2′-Me-G. For protein precipitation and extraction, each plasmasample (50 μL) was treated with 500 μL of 0.2% formic acid inacetonitrile and 20 μL of the internal standard working solution. Aftervortexing and centrifugation, 500 μL of the sample extracts weretransferred to a new plate, dried under N₂ at ˜28° C. and reconstitutedwith 75 μL of 0.2% FA in water. The extracts were chromatographed on anAquasil C18 column using a gradient system of 0.2% formic acid in waterand acetonitrile. The analytes were detected and quantified by tandemmass spectrometry in positive ion mode on an MDS Sciex API5000 equippedwith a Turbo Ionspray® interface. The calibration range was 0.500 (LLOQ)to 200 ng/mL in mouse plasma. The corresponding range for molar units is1.92 to 769 pmol/mL.

Liver samples were analyzed for the active species2′-methyl-2′-fluorouridine triphosphate (2′-Me-2′-F-U TP) by LC-MS/MS.2′-Me-2′-F-U TP levels were assayed by homogenizing (on ice) a knownweight of mouse liver with 4× volume of 0.95 M trichloroacetic acid(TCA). Internal standard solution was added to the homogenate followedby neutralization with 20% ammonium hydroxide solution and addition of500 μL 1% formic acid. The tissue samples were extracted by weak anionexchange solid phase extraction (SPE). Post extraction, the eluates wereevaporated under nitrogen, followed by reconstitution before injectiononto the LC-MS/MS system. The samples were chromatographed on a Luna NH2column using a gradient system of ammonium acetate (1 mM to 20 mM and pH8.0 to pH 10.0) in water and acetonitrile (70:30). The analyte wasdetected and quantified by tandem mass spectrometry in positive ion modeon an API4000 equipped with a Turbo Ionspray® interface. The calibrationrange was 10 to 10000 pmol/mL in mouse liver homogenate (50 to 50000pmol/g of mouse liver).

Results are provided in Table 5 below.

TABLE 4C Mouse plasma and liver pharmacokinetic parameters Compound(804b) Compound (804a) Diastereomer 1 Diastereomer 2 Cells Ia (R_(P)isomer) Ib (S_(P) isomer) (S_(P) isomer) (R_(P) isomer) Ms Plasma2′-Me-2′-F-U 480 260 320 320 AUC (pmol · hr/mL at 1 μmol/kg) Ms Liver2′-Me-2′-F-U TP 3200 430 250 310 AUC (pmol · hr/g at 1 μmol/kg)

Example 5 Pharmacokinetics of Liver Triphosphate and Plasma Prodrug andNucleoside Following a Single Oral Dose in Cynomolgus Monkeys

Abbreviations:

Mo=Monkey; TP=triphosphate; AUC=area under the concentration curve.

For each compound, a single oral dose at 10 mg/kg in PEG 200 (dosevolume 3 mL/kg) was administered to cynomolgus monkeys. Untreatedanimals were used for the collection of control liver. Plasma sampleswere collected at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours for compound 37,diastereomer 2. Terminal liver samples were collected from three animalsper time point at 6, 12 and 24 hours post dose for compound 37,diastereomer 2 and at 6 hours post dose for compound 44, diastereomer 2.Liver specimens were collected from all animals immediately after theincision. Freezing forceps stored in liquid nitrogen were used to freezethe liver before excision.

Plasma samples were analyzed for the prodrug and nucleoside by LC-MS/MS.For protein precipitation and extraction, each plasma sample (50 μL) wastreated with 500 μL of 0.2% formic acid in acetonitrile and 20 μL of anappropriate internal standard working solution. After vortexing andcentrifugation, 500 μL of the sample extracts were transferred to a newplate, dried under N₂ at ˜28° C. and reconstituted with 75 μL of 0.2% FAin water. The extracts were chromatographed on an Aquasil C18 columnusing a gradient system of 0.2% formic acid in water and acetonitrile.The analytes were detected and quantified by tandem mass spectrometry inpositive ion mode on an MDS Sciex API4000 equipped with a TurboIonspray® interface.

Liver samples were analyzed for the relevant nucleoside triphosphate byLC-MS/MS. The triphosphate levels were assayed by homogenizing (on ice)a known weight of liver with 4× volume of 0.95 M trichloroacetic acid(TCA). Appropriate internal standard solution was added to thehomogenate followed by neutralization with 20% ammonium hydroxidesolution and addition of 500 μL 1% formic acid. The tissue samples wereextracted by weak anion exchange solid phase extraction (SPE). Postextraction, the eluates were evaporated under nitrogen, followed byreconstitution before injection onto the LC-MS/MS system. The sampleswere chromatographed on a Luna NH₂ column using a gradient system ofammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water andacetonitrile (70:30). The analyte was detected and quantified by tandemmass spectrometry in positive ion mode on an API4000 equipped with aTurbo Ionspray® interface. Results are provided in Table 5 below.

TABLE 5 Pharmacokinetics of the prodrug and nucleoside in plasma andtriphosphate in liver of Cynomolgus monkeys Compound Compound 37Compound 40ii Compound 40i Diastereomer 2 Diastereomer 2 Diastereomer 1Dose (mg/kg) 10  10 10 Dose-normalized parameters^(a) Plasma prodrugC_(max) (pmol/mL) 840  ND^(b) T_(max) (hr) 4 ND AUC₀₋₂₄ 4000 ND (pmol ·hr/mL) Plasma nucleoside C_(max) (pmol/mL) 51 ND T_(max) (hr) 4 NDAUC₀₋₂₄ 650 ND (pmol · hr/mL) Nucleoside triphosphate in Liver C₆(pmol/g) 1500 120 270 C_(max) (pmol/g) 1700 ND T_(max) (hr) 12 NDAUC₀₋₂₄ (pmol · hr/g) 29000 ND ^(a)The C_(max), C₆ and AUC₀₋₂₄ data arenormalized to 1 μmol/kg dose ^(b)ND = not determined

Example 6 Hydrolysis of D-Alanine Prodrugs by Cathepsin A (CatA) and/orCarboxylesterase 1 (CES1) Introduction

The HCV NS5B RNA-dependent RNA polymerase is essential for the virallife cycle and thus, is a target for antiviral therapy. The active siteof NS5B is well conserved among the six genotypes of HCV and therefore,nucleos(t)ide analogs can act pan-genotypically. Furthermore, nucleotideinhibitors are typically not cross-resistant to other classes of directacting antivirals and can have a higher barrier to resistance comparedto non-nucleoside, protease and non-structural protein 5A (NS5A)inhibitors of HCV, making this class of HCV antivirals useful in a ofcombination HCV antiviral therapy.

Nucleoside analogs are typically competitive inhibitors of endogenousnucleosides and may act through chain termination upon incorporationinto the nascent HCV RNA chain during replication (Eldrup, et al. 2004,Structure-Activity Relationship of Purine Ribonucleosides for Inhibitionof Hepatitis C Virus RNA-Dependent RNA Polymerase. J. Med. Chem. 47:2283-2295). However, upon cell entry a nucleoside analog must first bephosphorylated to the active triphosphate species (Gardelli, et al 2009,Phosphoramidate prodrugs of 2′-C-methylcytidine for therapy of hepatitisC virus infection. J. Med. Chem. 52:5394-5407; Stein and Moore, 2001,Phosphorylation of nucleoside analog antiretrovirals: a review forclinicians. Pharmacotherapy 21:11-34; Tomassini, et al 2005, Inhibitoryeffect of 2′-substituted nucleosides on hepatitis C virus replicationcorrelates with metabolic properties in replicon cells. Antimicrob.Agents Chemother. 49:2050-2058; Murakami, et al 2007, Mechanism ofactivation of β-D-2′-deoxy-2′-fluoro-2′-C-methylcytidine and inhibitionof hepatitis C virus NS5B RNA polymerase. Antimicrob. Agents Chemother.51:503-509). A barrier to first generation nucleoside inhibitors was theoften inefficient conversion of the nucleoside to a nucleotidemonophosphate (NMP) by cellular kinases (Gardelli, et al 2009,Phosphoramidate prodrugs of 2′-C-methylcytidine for therapy of hepatitisC virus infection. J. Med. Chem. 52:5394-5407; Stein and Moore, 2001,Phosphorylation of nucleoside analog antiretrovirals: a review forclinicians. Pharmacotherapy 21:11-34; Murakami, et al 2007, Mechanism ofactivation of β-D-2′-deoxy-2′-fluoro-2′-C-methylcytidine and inhibitionof hepatitis C virus NS5B RNA polymerase. Antimicrob. Agents Chemother.51:503-509).

Second generation nucleoside analogs have been designed asliver-targeted nucleotide prodrugs, which bypass the rate-limiting NMPconversion to active species by delivering the nucleoside as amonophosphate prodrug. As GS-7977, Z4 and Z2 are pyrimidine nucleotideprodrugs that act by inhibition of the HCV NS5B RNA-dependent RNApolymerase through a 2′ modified UTP metabolite.

The intracellular metabolism (anabolism) of nucleotide analogs iscritical to their antiviral activity. A first step in the metabolism ofnucleotide prodrugs is the removal of the prodrug moiety by cellularenzymes followed by the activation of the nucleoside monophosphateanalog by host cell kinases for the sequential phosphorylation of theparent nucleos(t)ide analog to the 5′-triphosphate form, thebiologically active metabolite. Removal of the prodrug moiety ofteninvolves sequential or independent work of different cellular enzymes.

In vivo Z4 and Z2 appear to be effectively liver-targeted with a highliver:plasma ratio of drug metabolites. Both prodrugs are readilyconverted to the triphosphate (TP) metabolite in the liver of mice andmonkey producing more TP than GS-7977. The TP derivatives of Z4 and Z2selectively inhibit wild-type HCV NS5B enzyme in vitro withsubmicromolar IC₅₀ values. When tested in a genotype 1b HCVreplicon-bearing human hepatoma cell line (Huh-7), however, Z4 and Z2were largely inactive and failed to inhibit replicon reproduction(EC₅₀>50 μM). The in vitro antiviral inactivity of Z4 and Z2 is thoughtto reflect an inability of Huh-7 replicon cells to metabolize theprodrug moiety.

The first step of GS-7977 activation includes hydrolysis of the carboxylester by cathepsin A (CatA) and/or carboxylesterase 1 (CES1) (Saboulardet al, 2009, Characterization of the Activation Pathway ofPhosphoramidate Triester Prodrugs of Stavudine and Zidovudine. MolecularPharmacology. 56:693-704; Murakami et al, 2010, Mechanism of Activationof PSI-7851 and Its Diastereoisomer PSI-7977, JBC, 285(45):34337-34347;Sofia et al, 2010, Discovery of PSI-35366, a novel purine nucleotideprodrug for the treatment of hepatitis C virus. J Med Chem.53:7202-7218). Since CES1 is reported to be underexpressed in Huh-7replicon cells, CatA appears to be the major enzyme that hydrolyzesGS-7977 in these cells (Murakami et al, 2010, Mechanism of Activation ofPSI-7851 and Its Diastereoisomer PSI-7977, JBC, 285(45):34337-34347).

Methods

In this example the hydrolysis of the two D-ala-McGuigan prodrugs Z2(2′-Cl, 2′-MeUMP, diastereoisomer R_(P)) and Z4 (2′-F, 2′-MeUTP,diastereoisomer R_(P)) using CatA and CES1 was compared with activationof the L-ala-McGuigan prodrugs Y1 (2′-MeUTP, S_(P) stereoisomer),GS-7977 (X1, diastereoisomer S_(P)) and PSI-7976 (X2, diastereoisomerR_(P)).

CatA, cathepsin L (CatL) and CES1 were purchased from R & D Systems(Minneapolis, Minn.). Prior to the enzymatic hydrolysis reactions, CatAwas activated according to the manufacturer's instruction. Briefly, CatA(0.05 μg/μL) was incubated with CatL (0.005 μg/4) for 30 min at 37° C.in 25 mM MES pH 6.0 containing 5 mM DTT. The reaction was stopped byaddition of the CatL specific inhibitor E64 (10 μM).

The CatA assay was performed at 37° C. The reaction mixture contained 25mM MES buffer pH 6.0, 100 mM NaCl, 4 mM DTT and 100 μM of the compound.The reaction was started by addition of the activated CatA enzyme to afinal concentration of 0.005 μg/μL. One hundred-μL aliquots were takenafter 0.5 min, 3 hrs and 18 hrs of incubation. Reactions were stopped bymixing the sample with an equal volume of ice-cold methanol, and wereloaded on a HPLC for analysis.

CES1 assay was performed at 37° C. in the reaction mixture containing 50mM Tris/HCl buffer pH 7.5 and 100 μM of the compound. Reaction wasstarted by addition of the CES1 to the final concentration 0.01 μg/mL.100 μL aliquots were taken after 0.5 min, 3 hrs and 21 hrs of theincubation and the reaction was stopped by mixing with 100 μl of theice-cold methanol prior to HPLC analysis.

Samples were analyzed by HPLC using 5μ, C-18, 4.6×250 mm Phenomenex®Columbus column (Phenomenex USA, CA). The mobile phase consisted ofbuffer A (25 mM potassium phosphate with 5 mM tetrabutylammoniumdihydrogen phosphate pH 6.3) and buffer B (100% methanol). HPLC gradientconditions are shown in Table 6.

TABLE 6 Time (min) % A % B Flow (mL/min) 0 100 0 1 15 70 30 1 30 50 50 165 50 50 1 70 95 5 1

Results

As shown in Table 7, both CatA and CES1 hydrolyzed GS-7977 and itsdiastereoisomer PSI-7076. However, CatA cleaved GS-7977 (S_(P)configuration) 10 times more efficiently than its R_(P) diastereoisomer,while CES1 preferentially hydrolyzed the R_(P) diastereoisomer PSI-7976.These results are in good agreement with the literature (Murakami, et al2010, Mechanism of Activation of PSI-7851 and Its DiastereoisomerPSI-7977, JBC, 285(45):34337-34347).

TABLE 7 Huh-7 Reference EC₅₀, pmol * hr/ Liver TP Compound numberS_(P)/R_(P) (μM) 10⁶ cellsAUC₀₋₇₂ pmol * hr/g CatA CES1 GS-7977 X1 S_(P)0.25 63555 250 100% @ 12%/3 h;  L-Ala- 18 h 15%/21 h 2′F,2′MeUTPPSI-7976 X2 Rp 2.08 6527 310 10% 56%/3 h;  L-Ala- @18 h 94%/21 h2′F,2′MeUTP L-Ala- Y1 S_(P) 0.17 63740 420 100 @3 h Not tested 2′MeUTPD-Ala- Z1 S_(P) 7 4400 0% 4.5% @ 2′Cl,2′MeUTP 21 h Z2 R_(P) 5.9; 14; 47436.9 6200 0% 23% @ 3 h; 49% @ 21 h D-Ala- Z3 S_(P) 17 430 0% 0%2′F,2′MeUTP Z4 R_(P) >50 720.4 3200 0% 10% @ 3 h; 26% @ 21 h

In contrast, CatA was unable to hydrolyze any of the D-Ala-prodrugstested. However, both Z2 and Z4 were processed by CES1.

Since Huh-7 replicon-bearing cells have been found to express little orno CES1, CatA is the major enzyme that hydrolyzes GS-7977 in these cells(Murakami et al, 2010, Mechanism of Activation of PSI-7851 and ItsDiastereoisomer PSI-7977, JBC, 285(45):34337-34347). The inability ofCatA to activate the D-Ala-prodrugs Z2 and Z4 may explain the inactivityof these compounds in Huh-7 replicon-bearing cells, since the lack of invitro activity is believed to reflect low production of the active TPmoiety in Huh-7 replicon cells.

In vivo, high expression of CES1 in the liver coupled with highcatalytic efficiency and possible involvement of other liver enzymeappears to result in efficient conversion of Z2 and Z4 to theircorresponding triphosphate metabolites.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference. While theclaimed subject matter has been described in terms of variousembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the claimed subject matter is limited solely by the scope ofthe following claims, including equivalents thereof.

1-2. (canceled)
 3. A compound according to formula (II) or apharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof:

Base is guanine, uracil, cytosine, adenine or a derivative thereof; W isO; Y is —OR¹; R^(b1) is —CH₃; R^(b2) is —Cl; R¹ is phenyl; R¹⁰ is —CH₃R¹¹ is ethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl,cyclopentyl, or benzyl; R^(c) is —OH; R^(d) is —H; and R^(e) is —H.4-28. (canceled)
 29. The compound of claim 3, wherein said compound isaccording to the structure:


30. The compound of claim 3, wherein said compound is:


31. The compound of claim 3, wherein said compound is:


32. The compound of claim 3, wherein said compound is according to thestructure:


33. The compound according to claim 32, wherein said compound is:


34. The compound according to claim 32, wherein said compound is:


35. The compound according to claim 3, wherein said compound isaccording to the structure:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 36. The compound according to claim 35,wherein said compound is according to the structure:


37. The compound according to claim 35, wherein said compound is

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 38. The compound according to claim 37,wherein said compound is


39. A compound having the structure:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 40. A compound having the structure:


41. A nucleoside composition comprising a compound according to FormulaIIa or IIb:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof, wherein said composition is substantially freeof other stereoisomers of said compound: wherein Base is guanine,uracil, cytosine, adenine or a derivative thereof; W is O; Y is —OR¹; R¹is phenyl; R¹⁰ is —CH₃; R¹¹ is ethyl, propyl, isopropyl, n-propyl,butyl, n-butyl, t-butyl, or cyclopentyl; R^(b1) is —CH₃; R^(b2) is —Cl;R^(c) —OH; R^(d) is —H; and R^(e) is —H.
 42. The composition accordingto claim 41, wherein said compound is:


43. The composition according to claim 41, wherein said compound is:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 44. The composition according to claim 43,wherein said compound is:


45. A pharmaceutical composition comprising a compound according toformula (II) or a pharmaceutically acceptable salt, solvate, tautomericform or polymorphic form thereof:

wherein Base is guanine, uracil, cytosine, adenine or a derivativethereof; W is O; Y is —OR¹; R¹ is phenyl; R¹⁰ is —CH₃; R¹¹ is ethyl,propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl, or cyclopentyl;R^(b1) is —CH₃; R^(b2) is —Cl; R^(c) —OH; R^(d) is —H; and R^(e) is —H;and a pharmaceutically acceptable carrier.
 46. The pharmaceuticalcomposition of claim 45, wherein said composition provides atherapeutically effective amount of said compound for treating a humaninfected with HCV.
 47. The pharmaceutical composition of claim 46,wherein said compound is according to the structure:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 48. The pharmaceutical composition of claim47, wherein said compound is according to the structure:


49. The pharmaceutical composition of claim 46, wherein said compoundis:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 50. The pharmaceutical composition of claim49, wherein said compound is:


51. The pharmaceutical composition of claim 46, wherein said compoundis:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 52. The pharmaceutical composition of claim51, wherein said compound is:


53. The pharmaceutical composition of claim 51, wherein said compound isa designated enantiomer, and said composition is substantially free ofother stereoisomers of said compound.
 54. The pharmaceutical compositionof claim 52, wherein said compound is a designated enantiomer, and saidcomposition is substantially free of other stereoisomers of saidcompound.
 55. A method for the treatment of a human infected with ahepatitis C virus, comprising the administration to said human aneffective amount of a compound according to formula (II) or apharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof:

wherein Base is guanosine, uracil, cytosine, adenine or a derivativethereof; W is O; Y is —OR¹; R¹ is phenyl; R¹⁰ is —CH₃; R¹¹ is ethyl,propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl, or cyclopentyl;R^(b1) is —CH₃; R^(b2) is —Cl; R^(c) is —OH; R^(d) is —H; and R^(e) is—H.
 56. The method of claim 55, wherein said compound is according tothe structure:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 57. The method of claim 56, wherein saidcompound is according to the structure:


58. The method of claim 55, wherein said compound is:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 59. The method of claim 58, wherein saidcompound is:


60. The method of claim 55, wherein said compound is:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 61. The method of claim 60, wherein saidcompound is:


62. A method for the treatment of a human infected with a hepatitis Cvirus, comprising the administration to said human an effective amountof a pharmaceutical composition comprising a compound according toFormula IIa or IIb, or a pharmaceutically acceptable salt, solvate,tautomeric form or polymorphic form thereof:

wherein said composition is substantially free of other stereoisomers ofsaid compound: wherein Base is guanosine, uracil, cytosine, adenine or aderivative thereof; W is O; Y is —OR¹; R¹ is phenyl; R¹⁰ is —CH₃; R¹¹ isethyl, propyl, isopropyl, n-propyl, butyl, n-butyl, t-butyl, orcyclopentyl; R^(b1) is —CH₃; R^(b2) is —Cl; R^(c) —OH; R^(d) is —H; andR^(e) is —H; and a pharmaceutically acceptable carrier.
 63. The methodof claim 62, wherein said compound is:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 64. The method of claim 63, wherein saidcompound is:


65. The method of claim 62, wherein said compound is:

or a pharmaceutically acceptable salt, solvate, tautomeric form orpolymorphic form thereof.
 66. The method of claim 65, wherein saidcompound is: